Antisense modulation of hypothetical tumor endothelial marker expression

ABSTRACT

Antisense compounds, compositions and methods are provided for modulating the expression of hypothetical tumor endothelial marker. The compositions comprise antisense compounds, particularly antisense oligonucleotides, targeted to nucleic acids encoding hypothetical tumor endothelial marker. Methods of using these compounds for modulation of hypothetical tumor endothelial marker expression and for treatment of diseases associated with expression of hypothetical tumor endothelial marker are provided.

FIELD OF THE INVENTION

[0001] The present invention provides compositions and methods formodulating the expression of hypothetical tumor endothelial marker. Inparticular, this invention relates to compounds, particularlyoligonucleotides, specifically hybridizable with nucleic acids encodinghypothetical tumor endothelial marker. Such compounds have been shown tomodulate the expression of hypothetical tumor endothelial marker.

BACKGROUND OF THE INVENTION

[0002] Tumors require a blood supply for expansive growth, makinginhibition of tumor angiogenesis a very useful anticancer strategy.There are several advantages of targeting the endothelial cell whichline tumor vessels rather than the tumor cells themselves. Targetingendothelial cells rather than tumor cells obviates many of thepharmacokinetic problems associated with drug delivery. In addition, asignificant bystander effect can also be expected because eachendothelial cell supports the growth of many tumor cells and targetingthe genetically stable endothelial cells should reduce the likelihood ofdeveloping resistant disease and should be applicable to a wide varietyof tumor types (Carson-Walter et al., Cancer Res., 2001, 61, 6649-6655).

[0003] A comparison of gene expression patterns of endothelial cellsderived from blood vessels of normal and malignant colorectal tissueshas led to the finding that 46 of the 170 predominantly expressedtranscripts were elevated in tumor-associated endothelium (St Croix etal., Science, 2000, 289, 1197-1202). Several of these genes encodeextracellular matrix proteins but most are of unknown function (St Croixet al., Science, 2000, 289, 1197-1202).

[0004] The gene known as hypothetical tumor endothelial marker (alsoknown as CDNA FLJ10677 FIS, CLONE NT2RP2006467) is a gene of unknownfunction which shows a high degree of homology to tumor endothelialmarker 6 which was observed to exhibit a high tumor cell to normal celltag ratio in the aforementioned investigation (St Croix et al., Science,2000, 289, 1197-1202). The finding that hypothetical tumor endothelialmarker expression is significantly elevated in tumor cells indicatesthat modulation of its expression may prove a useful strategy forinhibition of tumor angiogenesis.

[0005] Currently, there are no known therapeutic agents that effectivelyinhibit the synthesis of hypothetical tumor endothelial marker.Consequently, there remains a long felt need for additional agentscapable of effectively inhibiting the function of hypothetical tumorendothelial marker.

[0006] Antisense technology is emerging as an effective means forreducing the expression of specific gene products and may thereforeprove to be uniquely useful in a number of therapeutic, diagnostic, andresearch applications for the modulation of expression of hypotheticaltumor endothelial marker.

[0007] The present invention provides compositions and methods formodulating expression of hypothetical tumor endothelial marker.

SUMMARY OF THE INVENTION

[0008] The present invention is directed to compounds, particularlyantisense oligonucleotides, which are targeted to a nucleic acidencoding hypothetical tumor endothelial marker, and which modulate theexpression of hypothetical tumor endothelial marker. Pharmaceutical andother compositions comprising the compounds of the invention are alsoprovided. Further provided are methods of modulating the expression ofhypothetical tumor endothelial marker in cells or tissues comprisingcontacting said cells or tissues with one or more of the antisensecompounds or compositions of the invention. Further provided are methodsof treating an animal, particularly a human, suspected of having orbeing prone to a disease or condition associated with expression ofhypothetical tumor endothelial marker by administering a therapeuticallyor prophylactically effective amount of one or more of the antisensecompounds or compositions of the invention.

DETAILED DESCRIPTION OF THE INVENTION

[0009] The present invention employs oligomeric compounds, particularlyantisense oligonucleotides, for use in modulating the function ofnucleic acid molecules encoding hypothetical tumor endothelial marker,ultimately modulating the amount of hypothetical tumor endothelialmarker produced. This is accomplished by providing antisense compoundswhich specifically hybridize with one or more nucleic acids encodinghypothetical tumor endothelial marker. As used herein, the terms “targetnucleic acid” and “nucleic acid encoding hypothetical tumor endothelialmarker” encompass DNA encoding hypothetical tumor endothelial marker,RNA (including pre-mRNA and mRNA) transcribed from such DNA, and alsocDNA derived from such RNA. The specific hybridization of an oligomericcompound with its target nucleic acid interferes with the normalfunction of the nucleic acid. This modulation of function of a targetnucleic acid by compounds which specifically hybridize to it isgenerally referred to as “antisense”. The functions of DNA to beinterfered with include replication and transcription. The functions ofRNA to be interfered with include all vital functions such as, forexample, translocation of the RNA to the site of protein translation,translocation of the RNA to sites within the cell which are distant fromthe site of RNA synthesis, translation of protein from the RNA, splicingof the RNA to yield one or more mRNA species, and catalytic activitywhich may be engaged in or facilitated by the RNA. The overall effect ofsuch interference with target nucleic acid function is modulation of theexpression of hypothetical tumor endothelial marker. In the context ofthe present invention, “modulation” means either an increase(stimulation) or a decrease (inhibition) in the expression of a gene. Inthe context of the present invention, inhibition is the preferred formof modulation of gene expression and mRNA is a preferred target.

[0010] It is preferred to target specific nucleic acids for antisense.“Targeting” an antisense compound to a particular nucleic acid, in thecontext of this invention, is a multistep process. The process usuallybegins with the identification of a nucleic acid sequence whose functionis to be modulated. This may be, for example, a cellular gene (or mRNAtranscribed from the gene) whose expression is associated with aparticular disorder or disease state, or a nucleic acid molecule from aninfectious agent. In the present invention, the target is a nucleic acidmolecule encoding hypothetical tumor endothelial marker. The targetingprocess also includes determination of a site or sites within this genefor the antisense interaction to occur such that the desired effect,e.g., detection or modulation of expression of the protein, will result.Within the context of the present invention, a preferred intragenic siteis the region encompassing the translation initiation or terminationcodon of the open reading frame (ORF) of the gene. Since, as is known inthe art, the translation initiation codon is typically 5′-AUG (intranscribed mRNA molecules; 5′-ATG in the corresponding DNA molecule),the translation initiation codon is also referred to as the “AUG codon,”the “start codon” or the “AUG start codon”. A minority of genes have atranslation initiation codon having the RNA sequence 5′-GUG, 5′-UUG or5′-CUG, and 5′-AUA, 5′-ACG and 5′-CUG have been shown to function invivo. Thus, the terms “translation initiation codon” and “start codon”can encompass many codon sequences, even though the initiator amino acidin each instance is typically methionine (in eukaryotes) orformylmethionine (in prokaryotes). It is also known in the art thateukaryotic and prokaryotic genes may have two or more alternative startcodons, any one of which may be preferentially utilized for translationinitiation in a particular cell type or tissue, or under a particularset of conditions. In the context of the invention, “start codon” and“translation initiation codon” refer to the codon or codons that areused in vivo to initiate translation of an mRNA molecule transcribedfrom a gene encoding hypothetical tumor endothelial marker, regardlessof the sequence(s) of such codons.

[0011] It is also known in the art that a translation termination codon(or “stop codon”) of a gene may have one of three sequences, i.e.,5′-UAA, 5′-UAG and 5′-UGA (the corresponding DNA sequences are 5′-TAA,5′-TAG and 5′-TGA, respectively). The terms “start codon region” and“translation initiation codon region” refer to a portion of such an mRNAor gene that encompasses from about 25 to about 50 contiguousnucleotides in either direction (i.e., 5′ or 3′) from a translationinitiation codon. Similarly, the terms “stop codon region” and“translation termination codon region” refer to a portion of such anmRNA or gene that encompasses from about 25 to about 50 contiguousnucleotides in either direction (i.e., 5′ or 3′) from a translationtermination codon.

[0012] The open reading frame (ORF) or “coding region,” which is knownin the art to refer to the region between the translation initiationcodon and the translation termination codon, is also a region which maybe targeted effectively. Other target regions include the 5′untranslated region (5′UTR), known in the art to refer to the portion ofan mRNA in the 5′ direction from the translation initiation codon, andthus including nucleotides between the 5′ cap site and the translationinitiation codon of an mRNA or corresponding nucleotides on the gene,and the 3′ untranslated region (3′UTR), known in the art to refer to theportion of an mRNA in the 3′ direction from the translation terminationcodon, and thus including nucleotides between the translationtermination codon and 3′ end of an mRNA or corresponding nucleotides onthe gene. The 5′ cap of an mRNA comprises an N7-methylated guanosineresidue joined to the 5′-most residue of the mRNA via a 5′-5′triphosphate linkage. The 5′ cap region of an mRNA is considered toinclude the 5′ cap structure itself as well as the first 50 nucleotidesadjacent to the cap. The 5′ cap region may also be a preferred targetregion.

[0013] Although some eukaryotic mRNA transcripts are directlytranslated, many contain one or more regions, known as “introns,” whichare excised from a transcript before it is translated. The remaining(and therefore translated) regions are known as “exons” and are splicedtogether to form a continuous mRNA sequence. mRNA splice sites, i.e.,intron-exon junctions, may also be preferred target regions, and areparticularly useful in situations where aberrant splicing is implicatedin disease, or where an overproduction of a particular mRNA spliceproduct is implicated in disease. Aberrant fusion junctions due torearrangements or deletions are also preferred targets. mRNA transcriptsproduced via the process of splicing of two (or more) mRNAs fromdifferent gene sources are known as “fusion transcripts”. It has alsobeen found that introns can be effective, and therefore preferred,target regions for antisense compounds targeted, for example, to DNA orpre-mRNA.

[0014] It is also known in the art that alternative RNA transcripts canbe produced from the same genomic region of DNA. These alternativetranscripts are generally known as “variants”. More specifically,“pre-mRNA variants” are transcripts produced from the same genomic DNAthat differ from other transcripts produced from the same genomic DNA ineither their start or stop position and contain both intronic andextronic regions.

[0015] Upon excision of one or more exon or intron regions or portionsthereof during splicing, pre-mRNA variants produce smaller “mRNAvariants”. Consequently, mRNA variants are processed pre-mRNA variantsand each unique pre-mRNA variant must always produce a unique mRNAvariant as a result of splicing. These mRNA variants are also known as“alternative splice variants”. If no splicing of the pre-mRNA variantoccurs then the pre-mRNA variant is identical to the mRNA variant.

[0016] It is also known in the art that variants can be produced throughthe use of alternative signals to start or stop transcription and thatpre-mRNAs and mRNAs can possess more that one start codon or stop codon.Variants that originate from a pre-mRNA or mRNA that use alternativestart codons are known as “alternative start variants” of that pre-mRNAor mRNA. Those transcripts that use an alternative stop codon are knownas “alternative stop variants” of that pre-mRNA or mRNA. One specifictype of alternative stop variant is the “polyA variant” in which themultiple transcripts produced result from the alternative selection ofone of the “polyA stop signals” by the transcription machinery, therebyproducing transcripts that terminate at unique polyA sites.

[0017] Once one or more target sites have been identified,oligonucleotides are chosen which are sufficiently complementary to thetarget, i.e., hybridize sufficiently well and with sufficientspecificity, to give the desired effect.

[0018] In the context of this invention, “hybridization” means hydrogenbonding, which may be Watson-Crick, Hoogsteen or reversed Hoogsteenhydrogen bonding, between complementary nucleoside or nucleotide bases.For example, adenine and thymine are complementary nucleobases whichpair through the formation of hydrogen bonds. “Complementary,” as usedherein, refers to the capacity for precise pairing between twonucleotides. For example, if a nucleotide at a certain position of anoligonucleotide is capable of hydrogen bonding with a nucleotide at thesame position of a DNA or RNA molecule, then the oligonucleotide and theDNA or RNA are considered to be complementary to each other at thatposition. The oligonucleotide and the DNA or RNA are complementary toeach other when a sufficient number of corresponding positions in eachmolecule are occupied by nucleotides which can hydrogen bond with eachother. Thus, “specifically hybridizable” and “complementary” are termswhich are used to indicate a sufficient degree of complementarity orprecise pairing such that stable and specific binding occurs between theoligonucleotide and the DNA or RNA target. It is understood in the artthat the sequence of an antisense compound need not be 100%complementary to that of its target nucleic acid to be specificallyhybridizable.

[0019] An antisense compound is specifically hybridizable when bindingof the compound to the target DNA or RNA molecule interferes with thenormal function of the target DNA or RNA to cause a loss of activity,and there is a sufficient degree of complementarity to avoidnon-specific binding of the antisense compound to non-target sequencesunder conditions in which specific binding is desired, i.e., underphysiological conditions in the case of in vivo assays or therapeutictreatment, and in the case of in vitro assays, under conditions in whichthe assays are performed. It is preferred that the antisense compoundsof the present invention comprise at least 80% sequence complementarityto a target region within the target nucleic acid, moreover that theycomprise 90% sequence complementarity and even more comprise 95%sequence complementarity to the target region within the target nucleicacid sequence to which they are targeted. For example, an antisensecompound in which 18 of 20 nucleobases of the antisense compound arecomplementary, and would therefore specifically hybridize, to a targetregion would represent 90 percent complementarity. Percentcomplementarity of an antisense compound with a region of a targetnucleic acid can be determined routinely using basic local alignmentsearch tools (BLAST programs) (Altschul et al., J. Mol. Biol., 1990,215, 403-410; Zhang and Madden, Genome Res., 1997, 7, 649-656).

[0020] Antisense and other compounds of the invention, which hybridizeto the target and inhibit expression of the target, are identifiedthrough experimentation, and representative sequences of these compoundsare hereinbelow identified as preferred embodiments of the invention.The sites to which these preferred antisense compounds are specificallyhybridizable are hereinbelow referred to as “preferred target regions”and are therefore preferred sites for targeting. As used herein the term“preferred target region” is defined as at least an 8-nucleobase portionof a target region to which an active antisense compound is targeted.While not wishing to be bound by theory, it is presently believed thatthese target regions represent regions of the target nucleic acid whichare accessible for hybridization.

[0021] While the specific sequences of particular preferred targetregions are set forth below, one of skill in the art will recognize thatthese serve to illustrate and describe particular embodiments within thescope of the present invention. Additional preferred target regions maybe identified by one having ordinary skill.

[0022] Target regions 8-80 nucleobases in length comprising a stretch ofat least eight (8) consecutive nucleobases selected from within theillustrative preferred target regions are considered to be suitablepreferred target regions as well.

[0023] Exemplary good preferred target regions include DNA or RNAsequences that comprise at least the 8 consecutive nucleobases from the5′-terminus of one of the illustrative preferred target regions (theremaining nucleobases being a consecutive stretch of the same DNA or RNAbeginning immediately upstream of the 5′-terminus of the target regionand continuing until the DNA or RNA contains about 8 to about 80nucleobases). Similarly good preferred target regions are represented byDNA or RNA sequences that comprise at least the 8 consecutivenucleobases from the 3′-terminus of one of the illustrative preferredtarget regions (the remaining nucleobases being a consecutive stretch ofthe same DNA or RNA beginning immediately downstream of the 3′-terminusof the target region and continuing until the DNA or RNA contains about8 to about 80 nucleobases). One having skill in the art, once armed withthe empirically-derived preferred target regions illustrated herein willbe able, without undue experimentation, to identify further preferredtarget regions. In addition, one having ordinary skill in the art willalso be able to identify additional compounds, including oligonucleotideprobes and primers, that specifically hybridize to these preferredtarget regions using techniques available to the ordinary practitionerin the art.

[0024] Antisense compounds are commonly used as research reagents anddiagnostics. For example, antisense oligonucleotides, which are able toinhibit gene expression with exquisite specificity, are often used bythose of ordinary skill to elucidate the function of particular genes.Antisense compounds are also used, for example, to distinguish betweenfunctions of various members of a biological pathway. Antisensemodulation has, therefore, been harnessed for research use.

[0025] For use in kits and diagnostics, the antisense compounds of thepresent invention, either alone or in combination with other antisensecompounds or therapeutics, can be used as tools in differential and/orcombinatorial analyses to elucidate expression patterns of a portion orthe entire complement of genes expressed within cells and tissues.

[0026] Expression patterns within cells or tissues treated with one ormore antisense compounds are compared to control cells or tissues nottreated with antisense compounds and the patterns produced are analyzedfor differential levels of gene expression as they pertain, for example,to disease association, signaling pathway, cellular localization,expression level, size, structure or function of the genes examined.These analyses can be performed on stimulated or unstimulated cells andin the presence or absence of other compounds which affect expressionpatterns.

[0027] Examples of methods of gene expression analysis known in the artinclude DNA arrays or microarrays (Brazma and Vilo, FEBS Lett., 2000,480, 17-24; Celis, et al., FEBS Lett., 2000, 480, 2-16), SAGE (serialanalysis of gene expression) (Madden, et al., Drug Discov. Today, 2000,5, 415-425), READS (restriction enzyme amplification of digested cDNAs)(Prashar and Weissman, Methods Enzymol., 1999, 303, 258-72), TOGA (totalgene expression analysis) (Sutcliffe, et al., Proc. Natl. Acad. Sci. U.S. A., 2000, 97, 1976-81), protein arrays and proteomics (Celis, et al.,FEBS Lett., 2000, 480, 2-16; Jungblut, et al., Electrophoresis, 1999,20, 2100-10), expressed sequence tag (EST) sequencing (Celis, et al.,FEBS Lett., 2000, 480, 2-16; Larsson, et al., J. Biotechnol., 2000, 80,143-57), subtractive RNA fingerprinting (SuRF) (Fuchs, et al., Anal.Biochem., 2000, 286, 91-98; Larson, et al., Cytometry, 2000, 41,203-208), subtractive cloning, differential display (DD) (Jurecic andBelmont, Curr. Opin. Microbiol., 2000, 3, 316-21), comparative genomichybridization (Carulli, et al., J. Cell Biochem. Suppl., 1998, 31,286-96), FISH (fluorescent in situ hybridization) techniques (Going andGusterson, Eur. J. Cancer, 1999, 35, 1895-904) and mass spectrometrymethods (reviewed in To, Comb. Chem. High Throughput Screen, 2000, 3,235-41).

[0028] The specificity and sensitivity of antisense is also harnessed bythose of skill in the art for therapeutic uses. Antisenseoligonucleotides have been employed as therapeutic moieties in thetreatment of disease states in animals and man. Antisenseoligonucleotide drugs, including ribozymes, have been safely andeffectively administered to humans and numerous clinical trials arepresently underway. It is thus established that oligonucleotides can beuseful therapeutic modalities that can be configured to be useful intreatment regimes for treatment of cells, tissues and animals,especially humans.

[0029] In the context of this invention, the term “oligonucleotide”refers to an oligomer or polymer of ribonucleic acid (RNA) ordeoxyribonucleic acid (DNA) or mimetics thereof. This term includesoligonucleotides composed of naturally-occurring nucleobases, sugars andcovalent internucleoside (backbone) linkages as well as oligonucleotideshaving non-naturally-occurring portions which function similarly. Suchmodified or substituted oligonucleotides are often preferred over nativeforms because of desirable properties such as, for example, enhancedcellular uptake, enhanced affinity for nucleic acid target and increasedstability in the presence of nucleases.

[0030] While antisense oligonucleotides are a preferred form ofantisense compound, the present invention comprehends other oligomericantisense compounds, including but not limited to oligonucleotidemimetics such as are described below. The antisense compounds inaccordance with this invention preferably comprise from about 8 to about80 nucleobases (i.e. from about 8 to about 80 linked nucleosides).Particularly preferred antisense compounds are antisenseoligonucleotides from about 8 to about 50 nucleobases, even morepreferably those comprising from about 12 to about 30 nucleobases.Antisense compounds include ribozymes, external guide sequence (EGS)oligonucleotides (oligozymes), and other short catalytic RNAs orcatalytic oligonucleotides which hybridize to the target nucleic acidand modulate its expression.

[0031] Antisense compounds 8-80 nucleobases in length comprising astretch of at least eight (8) consecutive nucleobases selected fromwithin the illustrative antisense compounds are considered to besuitable antisense compounds as well.

[0032] Exemplary preferred antisense compounds include DNA or RNAsequences that comprise at least the 8 consecutive nucleobases from the5′-terminus of one of the illustrative preferred antisense compounds(the remaining nucleobases being a consecutive stretch of the same DNAor RNA beginning immediately upstream of the 5′-terminus of theantisense compound which is specifically hybridizable to the targetnucleic acid and continuing until the DNA or RNA contains about 8 toabout 80 nucleobases). Similarly preferred antisense compounds arerepresented by DNA or RNA sequences that comprise at least the 8consecutive nucleobases from the 3′-terminus of one of the illustrativepreferred antisense compounds (the remaining nucleobases being aconsecutive stretch of the same DNA or RNA beginning immediatelydownstream of the 3′-terminus of the antisense compound which isspecifically hybridizable to the target nucleic acid and continuinguntil the DNA or RNA contains about 8 to about 80 nucleobases). Onehaving skill in the art, once armed with the empirically-derivedpreferred antisense compounds illustrated herein will be able, withoutundue experimentation, to identify further preferred antisensecompounds.

[0033] Antisense and other compounds of the invention, which hybridizeto the target and inhibit expression of the target, are identifiedthrough experimentation, and representative sequences of these compoundsare herein identified as preferred embodiments of the invention. Whilespecific sequences of the antisense compounds are set forth herein, oneof skill in the art will recognize that these serve to illustrate anddescribe particular embodiments within the scope of the presentinvention. Additional preferred antisense compounds may be identified byone having ordinary skill.

[0034] As is known in the art, a nucleoside is a base-sugar combination.The base portion of the nucleoside is normally a heterocyclic base. Thetwo most common classes of such heterocyclic bases are the purines andthe pyrimidines. Nucleotides are nucleosides that further include aphosphate group covalently linked to the sugar portion of thenucleoside. For those nucleosides that include a pentofuranosyl sugar,the phosphate group can be linked to either the 2′, 3′ or 5′ hydroxylmoiety of the sugar. In forming oligonucleotides, the phosphate groupscovalently link adjacent nucleosides to one another to form a linearpolymeric compound. In turn, the respective ends of this linearpolymeric structure can be further joined to form a circular structure,however, open linear structures are generally preferred. In addition,linear structures may also have internal nucleobase complementarity andmay therefore fold in a manner as to produce a double strandedstructure. Within the oligonucleotide structure, the phosphate groupsare commonly referred to as forming the internucleoside backbone of theoligonucleotide. The normal linkage or backbone of RNA and DNA is a 3′to 5′ phosphodiester linkage.

[0035] Specific examples of preferred antisense compounds useful in thisinvention include oligonucleotides containing modified backbones ornon-natural internucleoside linkages. As defined in this specification,oligonucleotides having modified backbones include those that retain aphosphorus atom in the backbone and those that do not have a phosphorusatom in the backbone. For the purposes of this specification, and assometimes referenced in the art, modified oligonucleotides that do nothave a phosphorus atom in their internucleoside backbone can also beconsidered to be oligonucleosides.

[0036] Preferred modified oligonucleotide backbones include, forexample, phosphorothioates, chiral phosphorothioates,phosphorodithioates, phosphotriesters, aminoalkylphosphotri-esters,methyl and other alkyl phosphonates including 3′-alkylene phosphonates,5′-alkylene phosphonates and chiral phosphonates, phosphinates,phosphoramidates including 3′-amino phosphoramidate andaminoalkylphosphoramidates, thionophosphoramidates,thionoalkylphosphonates, thionoalkylphosphotriesters, selenophosphatesand borano-phosphates having normal 3′-5′ linkages, 2′-5′ linked analogsof these, and those having inverted polarity wherein one or moreinternucleotide linkages is a 3′ to 3′, 5′ to 5′ or 2′ to 2′ linkage.Preferred oligonucleotides having inverted polarity comprise a single 3′to 3′ linkage at the 3′-most internucleotide linkage i.e. a singleinverted nucleoside residue which may be abasic (the nucleobase ismissing or has a hydroxyl group in place thereof). Various salts, mixedsalts and free acid forms are also included.

[0037] Representative United States patents that teach the preparationof the above phosphorus-containing linkages include, but are not limitedto, U.S. Pat. Nos.: 3,687,808; 4,469,863; 4,476,301; 5,023,243;5,177,196; 5,188,897; 5,264,423; 5,276,019; 5,278,302; 5,286,717;5,321,131; 5,399,676; 5,405,939; 5,453,496; 5,455,233; 5,466,677;5,476,925; 5,519,126; 5,536,821; 5,541,306; 5,550,111; 5,563,253;5,571,799; 5,587,361; 5,194,599; 5,565,555; 5,527,899; 5,721,218;5,672,697 and 5,625,050, certain of which are commonly owned with thisapplication, and each of which is herein incorporated by reference.

[0038] Preferred modified oligonucleotide backbones that do not includea phosphorus atom therein have backbones that are formed by short chainalkyl or cycloalkyl internucleoside linkages, mixed heteroatom and alkylor cycloalkyl internucleoside linkages, or one or more short chainheteroatomic or heterocyclic internucleoside linkages. These includethose having morpholino linkages (formed in part from the sugar portionof a nucleoside); siloxane backbones; sulfide, sulfoxide and sulfonebackbones; formacetyl and thioformacetyl backbones; methylene formacetyland thioformacetyl backbones; riboacetyl backbones; alkene containingbackbones; sulfamate backbones; methyleneimino and methylenehydrazinobackbones; sulfonate and sulfonamide backbones; amide backbones; andothers having mixed N, O, S and CH₂ component parts.

[0039] Representative United States patents that teach the preparationof the above oligonucleosides include, but are not limited to, U.S. Pat.Nos.: 5,034,506; 5,166,315; 5,185,444; 5,214,134; 5,216,141; 5,235,033;5,264,562; 5,264,564; 5,405,938; 5,434,257; 5,466,677; 5,470,967;5,489,677; 5,541,307; 5,561,225; 5,596,086; 5,602,240; 5,610,289;5,602,240; 5,608,046; 5,610,289; 5,618,704; 5,623,070; 5,663,312;5,633,360; 5,677,437; 5,792,608; 5,646,269 and 5,677,439, certain ofwhich are commonly owned with this application, and each of which isherein incorporated by reference.

[0040] In other preferred oligonucleotide mimetics, both the sugar andthe internucleoside linkage, i.e., the backbone, of the nucleotide unitsare replaced with novel groups. The base units are maintained forhybridization with an appropriate nucleic acid target compound. One sucholigomeric compound, an oligonucleotide mimetic that has been shown tohave excellent hybridization properties, is referred to as a peptidenucleic acid (PNA). In PNA compounds, the sugar-backbone of anoligonucleotide is replaced with an amide containing backbone, inparticular an aminoethylglycine backbone. The nucleobases are retainedand are bound directly or indirectly to aza nitrogen atoms of the amideportion of the backbone. Representative United States patents that teachthe preparation of PNA compounds include, but are not limited to, U.S.Pat. Nos.: 5,539,082; 5,714,331; and 5,719,262, each of which is hereinincorporated by reference. Further teaching of PNA compounds can befound in Nielsen et al., Science, 1991, 254, 1497-1500.

[0041] Most preferred embodiments of the invention are oligonucleotideswith phosphorothioate backbones and oligonucleosides with heteroatombackbones, and in particular —CH₂—NH—O—CH₂—, —CH₂—N(CH₃)—O—CH₂— [knownas a methylene (methylimino) or MMI backbone], —CH₂—O—N(CH₃)—CH₂—,—CH₂—N(CH₃)—N(CH₃)—CH₂— and —O—N(CH₃)—CH₂—CH₂— [wherein the nativephosphodiester backbone is represented as —O—P—O—CH₂—] of the abovereferenced U.S. Pat. No. 5,489,677, and the amide backbones of the abovereferenced U.S. Pat. No. 5,602,240. Also preferred are oligonucleotideshaving morpholino backbone structures of the above-referenced U.S. Pat.No. 5,034,506.

[0042] Modified oligonucleotides may also contain one or moresubstituted sugar moieties. Preferred oligonucleotides comprise one ofthe following at the 2′ position: OH; F; O—, S—, or N-alkyl; O—, S—, orN-alkenyl; O—, S— or N-alkynyl; or O-alkyl-O-alkyl, wherein the alkyl,alkenyl and alkynyl may be substituted or unsubstituted C₁ to C₁₀ alkylor C₂ to C₁₀ alkenyl and alkynyl. Particularly preferred areO[(CH₂)_(n)O]_(m)CH₃, O(CH₂)_(n)OCH₃, O(CH₂)_(n)NH₂, O(CH₂)_(n)CH₃,O(CH₂)_(n)ONH₂, and O(CH₂)_(n)ON[(CH₂)_(n)CH₃]₂, where n and m are from1 to about 10. Other preferred oligonucleotides comprise one of thefollowing at the 2′ position: C₁ to C₁₀ lower alkyl, substituted loweralkyl, alkenyl, alkynyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH,SCH₃, OCN, Cl, Br, CN, CF₃, OCF₃, SOCH₃, SO₂CH₃, ONO₂, NO₂, N₃, NH₂,heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino,substituted silyl, an RNA cleaving group, a reporter group, anintercalator, a group for improving the pharmacokinetic properties of anoligonucleotide, or a group for improving the pharmacodynamic propertiesof an oligonucleotide, and other substituents having similar properties.A preferred modification includes 2′-methoxyethoxy (2′-O—CH₂CH₂OCH₃,also known as 2′-O-(2-methoxyethyl) or 2′-MOE) (Martin et al., Helv.Chim. Acta, 1995, 78, 486-504) i.e., an alkoxyalkoxy group. A furtherpreferred modification includes 2′-dimethylaminooxyethoxy, i.e., aO(CH₂)₂ON(CH₃)₂ group, also known as 2′-DMAOE, as described in exampleshereinbelow, and 2′-dimethylaminoethoxyethoxy (also known in the art as2′-O-dimethyl-amino-ethoxy-ethyl or 2′-DMAEOE), i.e.,2′-O—CH₂—O—CH₂—N(CH₃)₂, also described in examples hereinbelow.

[0043] Other preferred modifications include 2′-methoxy (2′-O—CH₃),2′-aminopropoxy (2′-OCH₂CH₂CH₂NH₂), 2′-allyl (2′-CH₂—CH═CH₂), 2′-O-allyl(2′-O—CH₂—CH═CH₂) and 2′-fluoro (2′-F). The 2′-modification may be inthe arabino (up) position or ribo (down) position. A preferred2′-arabino modification is 2′-F. Similar modifications may also be madeat other positions on the oligonucleotide, particularly the 3′ positionof the sugar on the 3′ terminal nucleotide or in 2′-5′ linkedoligonucleotides and the 5′ position of 5′ terminal nucleotide.Oligonucleotides may also have sugar mimetics such as cyclobutylmoieties in place of the pentofuranosyl sugar. Representative UnitedStates patents that teach the preparation of such modified sugarstructures include, but are not limited to, U.S. Pat. Nos.: 4,981,957;5,118,800; 5,319,080; 5,359,044; 5,393,878; 5,446,137; 5,466,786;5,514,785; 5,519,134; 5,567,811; 5,576,427; 5,591,722; 5,597,909;5,610,300; 5,627,053; 5,639,873; 5,646,265; 5,658,873; 5,670,633;5,792,747; and 5,700,920, certain of which are commonly owned with theinstant application, and each of which is herein incorporated byreference in its entirety.

[0044] A further preferred modification includes Locked Nucleic Acids(LNAs) in which the 2′-hydroxyl group is linked to the 3′ or 4′ carbonatom of the sugar ring thereby forming a bicyclic sugar moiety. Thelinkage is preferably a methelyne (—CH₂—)_(n) group bridging the 2′oxygen atom and the 4′ carbon atom wherein n is 1 or 2. LNAs andpreparation thereof are described in WO 98/39352 and WO 99/14226.

[0045] Oligonucleotides may also include nucleobase (often referred toin the art simply as “base”) modifications or substitutions. As usedherein, “unmodified” or “natural” nucleobases include the purine basesadenine (A) and guanine (G), and the pyrimidine bases thymine (T),cytosine (C) and uracil (U). Modified nucleobases include othersynthetic and natural nucleobases such as 5-methylcytosine (5-me-C),5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine,6-methyl and other alkyl derivatives of adenine and guanine, 2-propyland other alkyl derivatives of adenine and guanine, 2-thiouracil,2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyl(—C≡C—CH₃) uracil and cytosine and other alkynyl derivatives ofpyrimidine bases, 6-azo uracil, cytosine and thymine, 5-uracil(pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl,8-hydroxyl and other 8-substituted adenines and guanines, 5-haloparticularly 5-bromo, 5-trifluoromethyl and other 5-substituted uracilsand cytosines, 7-methylguanine and 7-methyladenine, 2-F-adenine,2-amino-adenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and7-deazaadenine and 3-deazaguanine and 3-deazaadenine. Further modifiednucleobases include tricyclic pyrimidines such as phenoxazinecytidine(1H-pyrimido[5,4-b][1,4]benzoxazin-2(3H)-one), phenothiazinecytidine (1H-pyrimido[5,4-b][1,4]benzothiazin-2(3H)-one), G-clamps suchas a substituted phenoxazine cytidine (e.g.9-(2-aminoethoxy)-H-pyrimido[5,4-b][1,4]benzoxazin-2(3H)-one), carbazolecytidine (2H-pyrimido[4,5-b]indol-2-one), pyridoindole cytidine(H-pyrido[3′,2′:4,5]pyrrolo[2,3-d]pyrimidin-2-one). Modified nucleobasesmay also include those in which the purine or pyrimidine base isreplaced with other heterocycles, for example 7-deaza-adenine,7-deazaguanosine, 2-aminopyridine and 2-pyridone. Further nucleobasesinclude those disclosed in U.S. Pat. No. 3,687,808, those disclosed inThe Concise Encyclopedia Of Polymer Science And Engineering, pages858-859, Kroschwitz, J. I., ed. John Wiley & Sons, 1990, those disclosedby Englisch et al., Angewandte Chemie, International Edition, 1991, 30,613, and those disclosed by Sanghvi, Y. S., Chapter 15, AntisenseResearch and Applications, pages 289-302, Crooke, S. T. and Lebleu, B.ed., CRC Press, 1993. Certain of these nucleobases are particularlyuseful for increasing the binding affinity of the oligomeric compoundsof the invention. These include 5-substituted pyrimidines,6-azapyrimidines and N-2, N-6 and O-6 substituted purines, including2-aminopropyladenine, 5-propynyluracil and 5-propynylcytosine.5-methylcytosine substitutions have been shown to increase nucleic acidduplex stability by 0.6-1.2° C. (Sanghvi, Y. S., Crooke, S. T. andLebleu, B., eds., Antisense Research and Applications, CRC Press, BocaRaton, 1993, pp. 276-278) and are presently preferred basesubstitutions, even more particularly when combined with2′-O-methoxyethyl sugar modifications.

[0046] Representative United States patents that teach the preparationof certain of the above noted modified nucleobases as well as othermodified nucleobases include, but are not limited to, the above notedU.S. Pat. No. 3,687,808, as well as U.S. Pat. Nos.: 4,845,205;5,130,302; 5,134,066; 5,175,273; 5,367,066; 5,432,272; 5,457,187;5,459,255; 5,484,908; 5,502,177; 5,525,711; 5,552,540; 5,587,469;5,594,121, 5,596,091; 5,614,617; 5,645,985; 5,830,653; 5,763,588;6,005,096; and 5,681,941, certain of which are commonly owned with theinstant application, and each of which is herein incorporated byreference, and U.S. Pat. No. 5,750,692, which is commonly owned with theinstant application and also herein incorporated by reference.

[0047] Another modification of the oligonucleotides of the inventioninvolves chemically linking to the oligonucleotide one or more moietiesor conjugates which enhance the activity, cellular distribution orcellular uptake of the oligonucleotide. The compounds of the inventioncan include conjugate groups covalently bound to functional groups suchas primary or secondary hydroxyl groups. Conjugate groups of theinvention include intercalators, reporter molecules, polyamines,polyamides, polyethylene glycols, polyethers, groups that enhance thepharmacodynamic properties of oligomers, and groups that enhance thepharmacokinetic properties of oligomers. Typical conjugate groupsinclude cholesterols, lipids, phospholipids, biotin, phenazine, folate,phenanthridine, anthraquinone, acridine, fluores-ceins, rhodamines,coumarins, and dyes. Groups that enhance the pharmacodynamic properties,in the context of this invention, include groups that improve oligomeruptake, enhance oligomer resistance to degradation, and/or strengthensequence-specific hybridization with RNA. Groups that enhance thepharmacokinetic properties, in the context of this invention, includegroups that improve oligomer uptake, distribution, metabolism orexcretion. Representative conjugate groups are disclosed inInternational Patent Application PCT/US92/09196, filed Oct. 23, 1992 theentire disclosure of which is incorporated herein by reference.Conjugate moieties include but are not limited to lipid moieties such asa cholesterol moiety (Letsinger et al., Proc. Natl. Acad. Sci. USA,1989, 86, 6553-6556), cholic acid (Manoharan et al., Bioorg. Med. Chem.Let., 1994, 4, 1053-1060), a thioether, e.g., hexyl-S-tritylthiol(Manoharan et al., Ann. N.Y. Acad. Sci., 1992, 660, 306-309; Manoharanet al., Bioorg. Med. Chem. Let., 1993, 3, 2765-2770), a thiocholesterol(Oberhauser et al., Nucl. Acids Res., 1992, 20, 533-538), an aliphaticchain, e.g., dodecandiol or undecyl residues (Saison-Behmoaras et al.,EMBO J., 1991, 10, 1111-1118; Kabanov et al., FEBS Lett., 1990, 259,327-330; Svinarchuk et al., Biochimie, 1993, 75, 49-54), a phospholipid,e.g., di-hexadecyl-rac-glycerol or triethyl-ammonium1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate (Manoharan et al.,Tetrahedron Lett., 1995, 36, 3651-3654; Shea et al., Nucl. Acids Res.,1990, 18, 3777-3783), a polyamine or a polyethylene glycol chain(Manoharan et al., Nucleosides & Nucleotides, 1995, 14, 969-973), oradamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995, 36,3651-3654), a palmityl moiety (Mishra et al., Biochim. Biophys. Acta,1995, 1264, 229-237), or an octadecylamine orhexylamino-carbonyl-oxycholesterol moiety (Crooke et al., J. Pharmacol.Exp. Ther., 1996, 277, 923-937). Oligonucleotides of the invention mayalso be conjugated to active drug substances, for example, aspirin,warfarin, phenylbutazone, ibuprofen, suprofen, fenbufen, ketoprofen,(S)-(+)-pranoprofen, carprofen, dansylsarcosine, 2,3,5-triiodobenzoicacid, flufenamic acid, folinic acid, a benzothiadiazide, chlorothiazide,a diazepine, indomethicin, a barbiturate, a cephalosporin, a sulfa drug,an antidiabetic, an antibacterial or an antibiotic. Oligonucleotide-drugconjugates and their preparation are described in U.S. patentapplication Ser. No. 09/334,130 (filed Jun. 15, 1999) which isincorporated herein by reference in its entirety.

[0048] Representative United States patents that teach the preparationof such oligonucleotide conjugates include, but are not limited to, U.S.Pat. Nos.: 4,828,979; 4,948,882; 5,218,105; 5,525,465; 5,541,313;5,545,730; 5,552,538; 5,578,717, 5,580,731; 5,580,731; 5,591,584;5,109,124; 5,118,802; 5,138,045; 5,414,077; 5,486,603; 5,512,439;5,578,718; 5,608,046; 4,587,044; 4,605,735; 4,667,025; 4,762,779;4,789,737; 4,824,941; 4,835,263; 4,876,335; 4,904,582; 4,958,013;5,082,830; 5,112,963; 5,214,136; 5,082,830; 5,112,963; 5,214,136;5,245,022; 5,254,469; 5,258,506; 5,262,536; 5,272,250; 5,292,873;5,317,098; 5,371,241, 5,391,723; 5,416,203, 5,451,463; 5,510,475;5,512,667; 5,514,785; 5,565,552; 5,567,810; 5,574,142; 5,585,481;5,587,371; 5,595,726; 5,597,696; 5,599,923; 5,599,928 and 5,688,941,certain of which are commonly owned with the instant application, andeach of which is herein incorporated by reference.

[0049] It is not necessary for all positions in a given compound to beuniformly modified, and in fact more than one of the aforementionedmodifications may be incorporated in a single compound or even at asingle nucleoside within an oligonucleotide. The present invention alsoincludes antisense compounds which are chimeric compounds. “Chimeric”antisense compounds or “chimeras,” in the context of this invention, areantisense compounds, particularly oligonucleotides, which contain two ormore chemically distinct regions, each made up of at least one monomerunit, i.e., a nucleotide in the case of an oligonucleotide compound.These oligonucleotides typically contain at least one region wherein theoligonucleotide is modified so as to confer upon the oligonucleotideincreased resistance to nuclease degradation, increased cellular uptake,increased stability and/or increased binding affinity for the targetnucleic acid. An additional region of the oligonucleotide may serve as asubstrate for enzymes capable of cleaving RNA:DNA or RNA:RNA hybrids. Byway of example, RNAse H is a cellular endonuclease which cleaves the RNAstrand of an RNA:DNA duplex. Activation of RNase H, therefore, resultsin cleavage of the RNA target, thereby greatly enhancing the efficiencyof oligonucleotide inhibition of gene expression. The cleavage ofRNA:RNA hybrids can, in like fashion, be accomplished through theactions of endoribonucleases, such as interferon-induced RNAseL whichcleaves both cellular and viral RNA. Consequently, comparable resultscan often be obtained with shorter oligonucleotides when chimericoligonucleotides are used, compared to phosphorothioatedeoxyoligonucleotides hybridizing to the same target region. Cleavage ofthe RNA target can be routinely detected by gel electrophoresis and, ifnecessary, associated nucleic acid hybridization techniques known in theart.

[0050] Chimeric antisense compounds of the invention may be formed ascomposite structures of two or more oligonucleotides, modifiedoligonucleotides, oligonucleosides and/or oligonucleotide mimetics asdescribed above. Such compounds have also been referred to in the art ashybrids or gapmers. Representative United States patents that teach thepreparation of such hybrid structures include, but are not limited to,U.S. Pat. Nos.: 5,013,830; 5,149,797; 5,220,007; 5,256,775; 5,366,878;5,403,711; 5,491,133; 5,565,350; 5,623,065; 5,652,355; 5,652,356; and5,700,922, certain of which are commonly owned with the instantapplication, and each of which is herein incorporated by reference inits entirety.

[0051] The antisense compounds used in accordance with this inventionmay be conveniently and routinely made through the well-known techniqueof solid phase synthesis. Equipment for such synthesis is sold byseveral vendors including, for example, Applied Biosystems (Foster City,Calif.). Any other means for such synthesis known in the art mayadditionally or alternatively be employed. It is well known to usesimilar techniques to prepare oligonucleotides such as thephosphorothioates and alkylated derivatives.

[0052] The compounds of the invention may also be admixed, encapsulated,conjugated or otherwise associated with other molecules, moleculestructures or mixtures of compounds, as for example, liposomes,receptor-targeted molecules, oral, rectal, topical or otherformulations, for assisting in uptake, distribution and/or absorption.Representative United States patents that teach the preparation of suchuptake, distribution and/or absorption-assisting formulations include,but are not limited to, U.S. Pat. Nos.: 5,108,921; 5,354,844; 5,416,016;5,459,127; 5,521,291; 5,543,158; 5,547,932; 5,583,020; 5,591,721;4,426,330; 4,534,899; 5,013,556; 5,108,921; 5,213,804; 5,227,170;5,264,221; 5,356,633; 5,395,619; 5,416,016; 5,417,978; 5,462,854;5,469,854; 5,512,295; 5,527,528; 5,534,259; 5,543,152; 5,556,948;5,580,575; and 5,595,756, each of which is herein incorporated byreference.

[0053] The antisense compounds of the invention encompass anypharmaceutically acceptable salts, esters, or salts of such esters, orany other compound which, upon administration to an animal, including ahuman, is capable of providing (directly or indirectly) the biologicallyactive metabolite or residue thereof. Accordingly, for example, thedisclosure is also drawn to prodrugs and pharmaceutically acceptablesalts of the compounds of the invention, pharmaceutically acceptablesalts of such prodrugs, and other bioequivalents.

[0054] The term “prodrug” indicates a therapeutic agent that is preparedin an inactive form that is converted to an active form (i.e., drug)within the body or cells thereof by the action of endogenous enzymes orother chemicals and/or conditions. In particular, prodrug versions ofthe oligonucleotides of the invention are prepared as SATE[(S-acetyl-2-thioethyl)phosphate] derivatives according to the methodsdisclosed in WO 93/24510 to Gosselin et al., published Dec. 9, 1993 orin WO 94/26764 and U.S. Pat. No. 5,770,713 to Imbach et al.

[0055] The term “pharmaceutically acceptable salts” refers tophysiologically and pharmaceutically acceptable salts of the compoundsof the invention: i.e., salts that retain the desired biologicalactivity of the parent compound and do not impart undesiredtoxicological effects thereto.

[0056] Pharmaceutically acceptable base addition salts are formed withmetals or amines, such as alkali and alkaline earth metals or organicamines. Examples of metals used as cations are sodium, potassium,magnesium, calcium, and the like. Examples of suitable amines areN,N′-dibenzylethylenediamine, chloroprocaine, choline, diethanolamine,dicyclohexylamine, ethylenediamine, N-methylglucamine, and procaine(see, for example, Berge et al., “Pharmaceutical Salts,” J. of PharmaSci., 1977, 66, 1-19). The base addition salts of said acidic compoundsare prepared by contacting the free acid form with a sufficient amountof the desired base to produce the salt in the conventional manner. Thefree acid form may be regenerated by contacting the salt form with anacid and isolating the free acid in the conventional manner. The freeacid forms differ from their respective salt forms somewhat in certainphysical properties such as solubility in polar solvents, but otherwisethe salts are equivalent to their respective free acid for purposes ofthe present invention. As used herein, a “pharmaceutical addition salt”includes a pharmaceutically acceptable salt of an acid form of one ofthe components of the compositions of the invention. These includeorganic or inorganic acid salts of the amines. Preferred acid salts arethe hydrochlorides, acetates, salicylates, nitrates and phosphates.Other suitable pharmaceutically acceptable salts are well known to thoseskilled in the art and include basic salts of a variety of inorganic andorganic acids, such as, for example, with inorganic acids, such as forexample hydrochloric acid, hydrobromic acid, sulfuric acid or phosphoricacid; with organic carboxylic, sulfonic, sulfo or phospho acids orN-substituted sulfamic acids, for example acetic acid, propionic acid,glycolic acid, succinic acid, maleic acid, hydroxymaleic acid,methylmaleic acid, fumaric acid, malic acid, tartaric acid, lactic acid,oxalic acid, gluconic acid, glucaric acid, glucuronic acid, citric acid,benzoic acid, cinnamic acid, mandelic acid, salicylic acid,4-aminosalicylic acid, 2-phenoxybenzoic acid, 2-acetoxybenzoic acid,embonic acid, nicotinic acid or isonicotinic acid; and with amino acids,such as the 20 alpha-amino acids involved in the synthesis of proteinsin nature, for example glutamic acid or aspartic acid, and also withphenylacetic acid, methanesulfonic acid, ethanesulfonic acid,2-hydroxyethanesulfonic acid, ethane-1,2-disulfonic acid,benzenesulfonic acid, 4-methylbenzenesulfonic acid,naphthalene-2-sulfonic acid, naphthalene-1,5-disulfonic acid, 2- or3-phosphoglycerate, glucose-6-phosphate, N-cyclohexylsulfamic acid (withthe formation of cyclamates), or with other acid organic compounds, suchas ascorbic acid. Pharmaceutically acceptable salts of compounds mayalso be prepared with a pharmaceutically acceptable cation. Suitablepharmaceutically acceptable cations are well known to those skilled inthe art and include alkaline, alkaline earth, ammonium and quaternaryammonium cations. Carbonates or hydrogen carbonates are also possible.

[0057] For oligonucleotides, preferred examples of pharmaceuticallyacceptable salts include but are not limited to (a) salts formed withcations such as sodium, potassium, ammonium, magnesium, calcium,polyamines such as spermine and spermidine, etc.; (b) acid additionsalts formed with inorganic acids, for example hydrochloric acid,hydrobromic acid, sulfuric acid, phosphoric acid, nitric acid and thelike; (c) salts formed with organic acids such as, for example, aceticacid, oxalic acid, tartaric acid, succinic acid, maleic acid, fumaricacid, gluconic acid, citric acid, malic acid, ascorbic acid, benzoicacid, tannic acid, palmitic acid, alginic acid, polyglutamic acid,naphthalenesulfonic acid, methanesulfonic acid, p-toluenesulfonic acid,naphthalenedisulfonic acid, polygalacturonic acid, and the like; and (d)salts formed from elemental anions such as chlorine, bromine, andiodine.

[0058] The antisense compounds of the present invention can be utilizedfor diagnostics, therapeutics, prophylaxis and as research reagents andkits. For therapeutics, an animal, preferably a human, suspected ofhaving a disease or disorder which can be treated by modulating theexpression of hypothetical tumor endothelial marker is treated byadministering antisense compounds in accordance with this invention. Thecompounds of the invention can be utilized in pharmaceuticalcompositions by adding an effective amount of an antisense compound to asuitable pharmaceutically acceptable diluent or carrier. Use of theantisense compounds and methods of the invention may also be usefulprophylactically, e.g., to prevent or delay infection, inflammation ortumor formation, for example. The antisense compounds of the inventionare useful for research and diagnostics, because these compoundshybridize to nucleic acids encoding hypothetical tumor endothelialmarker, enabling sandwich and other assays to easily be constructed toexploit this fact. Hybridization of the antisense oligonucleotides ofthe invention with a nucleic acid encoding hypothetical tumorendothelial marker can be detected by means known in the art. Such meansmay include conjugation of an enzyme to the oligonucleotide,radiolabelling of the oligonucleotide or any other suitable detectionmeans. Kits using such detection means for detecting the level ofhypothetical tumor endothelial marker in a sample may also be prepared.

[0059] The present invention also includes pharmaceutical compositionsand formulations which include the antisense compounds of the invention.The pharmaceutical compositions of the present invention may beadministered in a number of ways depending upon whether local orsystemic treatment is desired and upon the area to be treated.Administration may be topical (including ophthalmic and to mucousmembranes including vaginal and rectal delivery), pulmonary, e.g., byinhalation or insufflation of powders or aerosols, including bynebulizer; intratracheal, intranasal, epidermal and transdermal), oralor parenteral. Parenteral administration includes intravenous,intraarterial, subcutaneous, intraperitoneal or intramuscular injectionor infusion; or intracranial, e.g., intrathecal or intraventricular,administration. Oligonucleotides with at least one 2′-O-methoxyethylmodification are believed to be particularly useful for oraladministration.

[0060] Pharmaceutical compositions and formulations for topicaladministration may include transdermal patches, ointments, lotions,creams, gels, drops, suppositories, sprays, liquids and powders.Conventional pharmaceutical carriers, aqueous, powder or oily bases,thickeners and the like may be necessary or desirable. Coated condoms,gloves and the like may also be useful. Preferred topical formulationsinclude those in which the oligonucleotides of the invention are inadmixture with a topical delivery agent such as lipids, liposomes, fattyacids, fatty acid esters, steroids, chelating agents and surfactants.Preferred lipids and liposomes include neutral (e.g.dioleoylphosphatidyl DOPE ethanolamine, dimyristoylphosphatidyl cholineDMPC, distearolyphosphatidyl choline) negative (e.g.dimyristoylphosphatidyl glycerol DMPG) and cationic (e.g.dioleoyltetramethylaminopropyl DOTAP and dioleoylphosphatidylethanolamine DOTMA). Oligonucleotides of the invention may beencapsulated within liposomes or may form complexes thereto, inparticular to cationic liposomes. Alternatively, oligonucleotides may becomplexed to lipids, in particular to cationic lipids. Preferred fattyacids and esters include but are not limited arachidonic acid, oleicacid, eicosanoic acid, lauric acid, caprylic acid, capric acid, myristicacid, palmitic acid, stearic acid, linoleic acid, linolenic acid,dicaprate, tricaprate, monoolein, dilaurin, glyceryl 1-monocaprate,1-dodecylazacycloheptan-2-one, an acylcarnitine, an acylcholine, or aC₁₋₁₀ alkyl ester (e.g. isopropylmyristate IPM), monoglyceride,diglyceride or pharmaceutically acceptable salt thereof. Topicalformulations are described in detail in U.S. patent application Ser. No.09/315,298 filed on May 20, 1999 which is incorporated herein byreference in its entirety.

[0061] Compositions and formulations for oral administration includepowders or granules, microparticulates, nanoparticulates, suspensions orsolutions in water or non-aqueous media, capsules, gel capsules,sachets, tablets or minitablets. Thickeners, flavoring agents, diluents,emulsifiers, dispersing aids or binders may be desirable. Preferred oralformulations are those in which oligonucleotides of the invention areadministered in conjunction with one or more penetration enhancerssurfactants and chelators. Preferred surfactants include fatty acidsand/or esters or salts thereof, bile acids and/or salts thereof.Preferred bile acids/salts include chenodeoxycholic acid (CDCA) andursodeoxychenodeoxycholic acid (UDCA), cholic acid, dehydrocholic acid,deoxycholic acid, glucholic acid, glycholic acid, glycodeoxycholic acid,taurocholic acid, taurodeoxycholic acid, sodiumtauro-24,25-dihydro-fusidate and sodium glycodihydrofusidate. Preferredfatty acids include arachidonic acid, undecanoic acid, oleic acid,lauric acid, caprylic acid, capric acid, myristic acid, palmitic acid,stearic acid, linoleic acid, linolenic acid, dicaprate, tricaprate,monoolein, dilaurin, glyceryl 1-monocaprate,1-dodecylazacycloheptan-2-one, an acylcarnitine, an acylcholine, or amonoglyceride, a diglyceride or a pharmaceutically acceptable saltthereof (e.g. sodium). Also preferred are combinations of penetrationenhancers, for example, fatty acids/salts in combination with bileacids/salts. A particularly preferred combination is the sodium salt oflauric acid, capric acid and UDCA. Further penetration enhancers includepolyoxyethylene-9-lauryl ether, polyoxyethylene-20-cetyl ether.Oligonucleotides of the invention may be delivered orally, in granularform including sprayed dried particles, or complexed to form micro ornanoparticles. Oligonucleotide complexing agents include poly-aminoacids; polyimines; polyacrylates; polyalkylacrylates, polyoxethanes,polyalkylcyanoacrylates; cationized gelatins, albumins, starches,acrylates, polyethyleneglycols (PEG) and starches;polyalkylcyanoacrylates; DEAE-derivatized polyimines, pollulans,celluloses and starches. Particularly preferred complexing agentsinclude chitosan, N-trimethylchitosan, poly-L-lysine, polyhistidine,polyornithine, polyspermines, protamine, polyvinylpyridine,polythiodiethylamino-methylethylene P(TDAE), polyaminostyrene (e.g.p-amino), poly(methylcyanoacrylate), poly(ethylcyanoacrylate),poly(butylcyanoacrylate), poly(isobutylcyanoacrylate),poly(isohexylcynaoacrylate), DEAE-methacrylate, DEAE-hexylacrylate,DEAE-acrylamide, DEAE-albumin and DEAE-dextran, polymethylacrylate,polyhexylacrylate, poly(D,L-lactic acid), poly(DL-lactic-co-glycolicacid (PLGA), alginate, and polyethyleneglycol (PEG). Oral formulationsfor oligonucleotides and their preparation are described in detail inU.S. application Ser. Nos. 08/886,829 (filed Jul. 1, 1997), 09/108,673(filed Jul. 1, 1998), 09/256,515 (filed Feb. 23, 1999), 09/082,624(filed May 21, 1998) and 09/315,298 (filed May 20, 1999), each of whichis incorporated herein by reference in their entirety.

[0062] Compositions and formulations for parenteral, intrathecal orintraventricular administration may include sterile aqueous solutionswhich may also contain buffers, diluents and other suitable additivessuch as, but not limited to, penetration enhancers, carrier compoundsand other pharmaceutically acceptable carriers or excipients.

[0063] Pharmaceutical compositions of the present invention include, butare not limited to, solutions, emulsions, and liposome-containingformulations. These compositions may be generated from a variety ofcomponents that include, but are not limited to, preformed liquids,self-emulsifying solids and self-emulsifying semisolids.

[0064] The pharmaceutical formulations of the present invention, whichmay conveniently be presented in unit dosage form, may be preparedaccording to conventional techniques well known in the pharmaceuticalindustry. Such techniques include the step of bringing into associationthe active ingredients with the pharmaceutical carrier(s) orexcipient(s). In general, the formulations are prepared by uniformly andintimately bringing into association the active ingredients with liquidcarriers or finely divided solid carriers or both, and then, ifnecessary, shaping the product.

[0065] The compositions of the present invention may be formulated intoany of many possible dosage forms such as, but not limited to, tablets,capsules, gel capsules, liquid syrups, soft gels, suppositories, andenemas. The compositions of the present invention may also be formulatedas suspensions in aqueous, non-aqueous or mixed media. Aqueoussuspensions may further contain substances which increase the viscosityof the suspension including, for example, sodium carboxymethylcellulose,sorbitol and/or dextran. The suspension may also contain stabilizers.

[0066] In one embodiment of the present invention the pharmaceuticalcompositions may be formulated and used as foams. Pharmaceutical foamsinclude formulations such as, but not limited to, emulsions,microemulsions, creams, jellies and liposomes. While basically similarin nature these formulations vary in the components and the consistencyof the final product. The preparation of such compositions andformulations is generally known to those skilled in the pharmaceuticaland formulation arts and may be applied to the formulation of thecompositions of the present invention.

[0067] Emulsions

[0068] The compositions of the present invention may be prepared andformulated as emulsions. Emulsions are typically heterogenous systems ofone liquid dispersed in another in the form of droplets usuallyexceeding 0.1 μm in diameter (Idson, in Pharmaceutical Dosage Forms,Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., NewYork, N.Y., volume 1, p. 199; Rosoff, in Pharmaceutical Dosage Forms,Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., NewYork, N.Y., Volume 1, p. 245; Block in Pharmaceutical Dosage Forms,Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., NewYork, N.Y., volume 2, p. 335; Higuchi et al., in Remington'sPharmaceutical Sciences, Mack Publishing Co., Easton, Pa., 1985, p.301). Emulsions are often biphasic systems comprising two immiscibleliquid phases intimately mixed and dispersed with each other. Ingeneral, emulsions may be of either the water-in-oil (w/o) or theoil-in-water (o/w) variety. When an aqueous phase is finely divided intoand dispersed as minute droplets into a bulk oily phase, the resultingcomposition is called a water-in-oil (w/o) emulsion. Alternatively, whenan oily phase is finely divided into and dispersed as minute dropletsinto a bulk aqueous phase, the resulting composition is called anoil-in-water (o/w) emulsion. Emulsions may contain additional componentsin addition to the dispersed phases, and the active drug which may bepresent as a solution in either the aqueous phase, oily phase or itselfas a separate phase. Pharmaceutical excipients such as emulsifiers,stabilizers, dyes, and anti-oxidants may also be present in emulsions asneeded. Pharmaceutical emulsions may also be multiple emulsions that arecomprised of more than two phases such as, for example, in the case ofoil-in-water-in-oil (o/w/o) and water-in-oil-in-water (w/o/w) emulsions.Such complex formulations often provide certain advantages that simplebinary emulsions do not. Multiple emulsions in which individual oildroplets of an o/w emulsion enclose small water droplets constitute aw/o/w emulsion. Likewise a system of oil droplets enclosed in globulesof water stabilized in an oily continuous phase provides an o/w/oemulsion.

[0069] Emulsions are characterized by little or no thermodynamicstability. Often, the dispersed or discontinuous phase of the emulsionis well dispersed into the external or continuous phase and maintainedin this form through the means of emulsifiers or the viscosity of theformulation. Either of the phases of the emulsion may be a semisolid ora solid, as is the case of emulsion-style ointment bases and creams.Other means of stabilizing emulsions entail the use of emulsifiers thatmay be incorporated into either phase of the emulsion. Emulsifiers maybroadly be classified into four categories: synthetic surfactants,naturally occurring emulsifiers, absorption bases, and finely dispersedsolids (Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger andBanker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p.199).

[0070] Synthetic surfactants, also known as surface active agents, havefound wide applicability in the formulation of emulsions and have beenreviewed in the literature (Rieger, in Pharmaceutical Dosage Forms,Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., NewYork, N.Y., volume 1, p. 285; Idson, in Pharmaceutical Dosage Forms,Lieberman, Rieger and Banker (Eds.), Marcel Dekker, Inc., New York,N.Y., 1988, volume 1, p. 199). Surfactants are typically amphiphilic andcomprise a hydrophilic and a hydrophobic portion. The ratio of thehydrophilic to the hydrophobic nature of the surfactant has been termedthe hydrophile/lipophile balance (HLB) and is a valuable tool incategorizing and selecting surfactants in the preparation offormulations. Surfactants may be classified into different classes basedon the nature of the hydrophilic group: nonionic, anionic, cationic andamphoteric (Rieger, in Pharmaceutical Dosage Forms, Lieberman, Riegerand Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1,p. 285).

[0071] Naturally occurring emulsifiers used in emulsion formulationsinclude lanolin, beeswax, phosphatides, lecithin and acacia. Absorptionbases possess hydrophilic properties such that they can soak up water toform w/o emulsions yet retain their semisolid consistencies, such asanhydrous lanolin and hydrophilic petrolatum. Finely divided solids havealso been used as good emulsifiers especially in combination withsurfactants and in viscous preparations. These include polar inorganicsolids, such as heavy metal hydroxides, nonswelling clays such asbentonite, attapulgite, hectorite, kaolin, montmorillonite, colloidalaluminum silicate and colloidal magnesium aluminum silicate, pigmentsand nonpolar solids such as carbon or glyceryl tristearate.

[0072] A large variety of non-emulsifying materials are also included inemulsion formulations and contribute to the properties of emulsions.These include fats, oils, waxes, fatty acids, fatty alcohols, fattyesters, humectants, hydrophilic colloids, preservatives and antioxidants(Block, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker(Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 335;Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker(Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199).

[0073] Hydrophilic colloids or hydrocolloids include naturally occurringgums and synthetic polymers such as polysaccharides (for example,acacia, agar, alginic acid, carrageenan, guar gum, karaya gum, andtragacanth), cellulose derivatives (for example, carboxymethylcelluloseand carboxypropylcellulose), and synthetic polymers (for example,carbomers, cellulose ethers, and carboxyvinyl polymers). These disperseor swell in water to form colloidal solutions that stabilize emulsionsby forming strong interfacial films around the dispersed-phase dropletsand by increasing the viscosity of the external phase.

[0074] Since emulsions often contain a number of ingredients such ascarbohydrates, proteins, sterols and phosphatides that may readilysupport the growth of microbes, these formulations often incorporatepreservatives. Commonly used preservatives included in emulsionformulations include methyl paraben, propyl paraben, quaternary ammoniumsalts, benzalkonium chloride, esters of p-hydroxybenzoic acid, and boricacid. Antioxidants are also commonly added to emulsion formulations toprevent deterioration of the formulation. Antioxidants used may be freeradical scavengers such as tocopherols, alkyl gallates, butylatedhydroxyanisole, butylated hydroxytoluene, or reducing agents such asascorbic acid and sodium metabisulfite, and antioxidant synergists suchas citric acid, tartaric acid, and lecithin.

[0075] The application of emulsion formulations via dermatological, oraland parenteral routes and methods for their manufacture have beenreviewed in the literature (Idson, in Pharmaceutical Dosage Forms,Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., NewYork, N.Y., volume 1, p. 199). Emulsion formulations for oral deliveryhave been very widely used because of ease of formulation, as well asefficacy from an absorption and bioavailability standpoint (Rosoff, inPharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988,Marcel Dekker, Inc., New York, N.Y., volume 1, p. 245; Idson, inPharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988,Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199). Mineral-oil baselaxatives, oil-soluble vitamins and high fat nutritive preparations areamong the materials that have commonly been administered orally as o/wemulsions.

[0076] In one embodiment of the present invention, the compositions ofoligonucleotides and nucleic acids are formulated as microemulsions. Amicroemulsion may be defined as a system of water, oil and amphiphilewhich is a single optically isotropic and thermodynamically stableliquid solution (Rosoff, in Pharmaceutical Dosage Forms, Lieberman,Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y.,volume 1, p. 245). Typically microemulsions are systems that areprepared by first dispersing an oil in an aqueous surfactant solutionand then adding a sufficient amount of a fourth component, generally anintermediate chain-length alcohol to form a transparent system.Therefore, microemulsions have also been described as thermodynamicallystable, isotropically clear dispersions of two immiscible liquids thatare stabilized by interfacial films of surface-active molecules (Leungand Shah, in: Controlled Release of Drugs: Polymers and AggregateSystems, Rosoff, M., Ed., 1989, VCH Publishers, New York, pages185-215). Microemulsions commonly are prepared via a combination ofthree to five components that include oil, water, surfactant,cosurfactant and electrolyte. Whether the microemulsion is of thewater-in-oil (w/o) or an oil-in-water (o/w) type is dependent on theproperties of the oil and surfactant used and on the structure andgeometric packing of the polar heads and hydrocarbon tails of thesurfactant molecules (Schott, in Remington's Pharmaceutical Sciences,Mack Publishing Co., Easton, Pa., 1985, p. 271).

[0077] The phenomenological approach utilizing phase diagrams has beenextensively studied and has yielded a comprehensive knowledge, to oneskilled in the art, of how to formulate microemulsions (Rosoff, inPharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988,Marcel Dekker, Inc., New York, N.Y., volume 1, p. 245; Block, inPharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988,Marcel Dekker, Inc., New York, N.Y., volume 1, p. 335). Compared toconventional emulsions, microemulsions offer the advantage ofsolubilizing water-insoluble drugs in a formulation of thermodynamicallystable droplets that are formed spontaneously.

[0078] Surfactants used in the preparation of microemulsions include,but are not limited to, ionic surfactants, non-ionic surfactants, Brij96, polyoxyethylene oleyl ethers, polyglycerol fatty acid esters,tetraglycerol monolaurate (ML310), tetraglycerol monooleate (MO310),hexaglycerol monooleate (PO310), hexaglycerol pentaoleate (PO500),decaglycerol monocaprate (MCA750), decaglycerol monooleate (MO750),decaglycerol sequioleate (SO750), decaglycerol decaoleate (DA0750),alone or in combination with cosurfactants. The cosurfactant, usually ashort-chain alcohol such as ethanol, 1-propanol, and 1-butanol, servesto increase the interfacial fluidity by penetrating into the surfactantfilm and consequently creating a disordered film because of the voidspace generated among surfactant molecules. Microemulsions may, however,be prepared without the use of cosurfactants and alcohol-freeself-emulsifying microemulsion systems are known in the art. The aqueousphase may typically be, but is not limited to, water, an aqueoussolution of the drug, glycerol, PEG300, PEG400, polyglycerols, propyleneglycols, and derivatives of ethylene glycol. The oil phase may include,but is not limited to, materials such as Captex 300, Captex 355, CapmulMCM, fatty acid esters, medium chain (C8-C12) mono, di, andtri-glycerides, polyoxyethylated glyceryl fatty acid esters, fattyalcohols, polyglycolized glycerides, saturated polyglycolized C₈-C₁₀glycerides, vegetable oils and silicone oil.

[0079] Microemulsions are particularly of interest from the standpointof drug solubilization and the enhanced absorption of drugs. Lipid basedmicroemulsions (both o/w and w/o) have been proposed to enhance the oralbioavailability of drugs, including peptides (Constantinides et al.,Pharmaceutical Research, 1994, 11, 1385-1390; Ritschel, Meth. Find. Exp.Clin. Pharmacol., 1993, 13, 205). Microemulsions afford advantages ofimproved drug solubilization, protection of drug from enzymatichydrolysis, possible enhancement of drug absorption due tosurfactant-induced alterations in membrane fluidity and permeability,ease of preparation, ease of oral administration over solid dosageforms, improved clinical potency, and decreased toxicity (Constantinideset al., Pharmaceutical Research, 1994, 11, 1385; Ho et al., J. Pharm.Sci., 1996, 85, 138-143). Often microemulsions may form spontaneouslywhen their components are brought together at ambient temperature. Thismay be particularly advantageous when formulating thermolabile drugs,peptides or oligonucleotides. Microemulsions have also been effective inthe transdermal delivery of active components in both cosmetic andpharmaceutical applications. It is expected that the microemulsioncompositions and formulations of the present invention will facilitatethe increased systemic absorption of oligonucleotides and nucleic acidsfrom the gastrointestinal tract, as well as improve the local cellularuptake of oligonucleotides and nucleic acids within the gastrointestinaltract, vagina, buccal cavity and other areas of administration.

[0080] Microemulsions of the present invention may also containadditional components and additives such as sorbitan monostearate (Grill3), Labrasol, and penetration enhancers to improve the properties of theformulation and to enhance the absorption of the oligonucleotides andnucleic acids of the present invention. Penetration enhancers used inthe microemulsions of the present invention may be classified asbelonging to one of five broad categories—surfactants, fatty acids, bilesalts, chelating agents, and non-chelating non-surfactants (Lee et al.,Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p. 92). Eachof these classes has been discussed above.

[0081] Liposomes

[0082] There are many organized surfactant structures besidesmicroemulsions that have been studied and used for the formulation ofdrugs. These include monolayers, micelles, bilayers and vesicles.Vesicles, such as liposomes, have attracted great interest because oftheir specificity and the duration of action they offer from thestandpoint of drug delivery. As used in the present invention, the term“liposome” means a vesicle composed of amphiphilic lipids arranged in aspherical bilayer or bilayers.

[0083] Liposomes are unilamellar or multilamellar vesicles which have amembrane formed from a lipophilic material and an aqueous interior. Theaqueous portion contains the composition to be delivered. Cationicliposomes possess the advantage of being able to fuse to the cell wall.Non-cationic liposomes, although not able to fuse as efficiently withthe cell wall, are taken up by macrophages in vivo.

[0084] In order to cross intact mammalian skin, lipid vesicles must passthrough a series of fine pores, each with a diameter less than 50 nm,under the influence of a suitable transdermal gradient. Therefore, it isdesirable to use a liposome which is highly deformable and able to passthrough such fine pores.

[0085] Further advantages of liposomes include; liposomes obtained fromnatural phospholipids are biocompatible and biodegradable; liposomes canincorporate a wide range of water and lipid soluble drugs; liposomes canprotect encapsulated drugs in their internal compartments frommetabolism and degradation (Rosoff, in Pharmaceutical Dosage Forms,Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., NewYork, N.Y., volume 1, p. 245). Important considerations in thepreparation of liposome formulations are the lipid surface charge,vesicle size and the aqueous volume of the liposomes.

[0086] Liposomes are useful for the transfer and delivery of activeingredients to the site of action. Because the liposomal membrane isstructurally similar to biological membranes, when liposomes are appliedto a tissue, the liposomes start to merge with the cellular membranesand as the merging of the liposome and cell progresses, the liposomalcontents are emptied into the cell where the active agent may act.

[0087] Liposomal formulations have been the focus of extensiveinvestigation as the mode of delivery for many drugs. There is growingevidence that for topical administration, liposomes present severaladvantages over other formulations. Such advantages include reducedside-effects related to high systemic absorption of the administereddrug, increased accumulation of the administered drug at the desiredtarget, and the ability to administer a wide variety of drugs, bothhydrophilic and hydrophobic, into the skin.

[0088] Several reports have detailed the ability of liposomes to deliveragents including high-molecular weight DNA into the skin. Compoundsincluding analgesics, antibodies, hormones and high-molecular weightDNAs have been administered to the skin. The majority of applicationsresulted in the targeting of the upper epidermis.

[0089] Liposomes fall into two broad classes. Cationic liposomes arepositively charged liposomes which interact with the negatively chargedDNA molecules to form a stable complex. The positively chargedDNA/liposome complex binds to the negatively charged cell surface and isinternalized in an endosome. Due to the acidic pH within the endosome,the liposomes are ruptured, releasing their contents into the cellcytoplasm (Wang et al., Biochem. Biophys. Res. Commun., 1987, 147,980-985).

[0090] Liposomes which are pH-sensitive or negatively-charged, entrapDNA rather than complex with it. Since both the DNA and the lipid aresimilarly charged, repulsion rather than complex formation occurs.Nevertheless, some DNA is entrapped within the aqueous interior of theseliposomes. pH-sensitive liposomes have been used to deliver DNA encodingthe thymidine kinase gene to cell monolayers in culture. Expression ofthe exogenous gene was detected in the target cells (Zhou et al.,Journal of Controlled Release, 1992, 19, 269-274).

[0091] One major type of liposomal composition includes phospholipidsother than naturally-derived phosphatidylcholine. Neutral liposomecompositions, for example, can be formed from dimyristoylphosphatidylcholine (DMPC) or dipalmitoyl phosphatidylcholine (DPPC).Anionic liposome compositions generally are formed from dimyristoylphosphatidylglycerol, while anionic fusogenic liposomes are formedprimarily from dioleoyl phosphatidylethanolamine (DOPE). Another type ofliposomal composition is formed from phosphatidylcholine (PC) such as,for example, soybean PC, and egg PC. Another type is formed frommixtures of phospholipid and/or phosphatidylcholine and/or cholesterol.

[0092] Several studies have assessed the topical delivery of liposomaldrug formulations to the skin. Application of liposomes containinginterferon to guinea pig skin resulted in a reduction of skin herpessores while delivery of interferon via other means (e.g. as a solutionor as an emulsion) were ineffective (Weiner et al., Journal of DrugTargeting, 1992, 2, 405-410). Further, an additional study tested theefficacy of interferon administered as part of a liposomal formulationto the administration of interferon using an aqueous system, andconcluded that the liposomal formulation was superior to aqueousadministration (du Plessis et al., Antiviral Research, 1992, 18,259-265).

[0093] Non-ionic liposomal systems have also been examined to determinetheir utility in the delivery of drugs to the skin, in particularsystems comprising non-ionic surfactant and cholesterol. Non-ionicliposomal formulations comprising Novasome™ I (glyceryldilaurate/cholesterol/polyoxyethylene-10-stearyl ether) and Novasome™ II(glyceryl distearate/cholesterol/polyoxyethylene-10-stearyl ether) wereused to deliver cyclosporin-A into the dermis of mouse skin. Resultsindicated that such non-ionic liposomal systems were effective infacilitating the deposition of cyclosporin-A into different layers ofthe skin (Hu et al. S.T.P.Pharma. Sci., 1994, 4, 6, 466).

[0094] Liposomes also include “sterically stabilized” liposomes, a termwhich, as used herein, refers to liposomes comprising one or morespecialized lipids that, when incorporated into liposomes, result inenhanced circulation lifetimes relative to liposomes lacking suchspecialized lipids. Examples of sterically stabilized liposomes arethose in which part of the vesicle-forming lipid portion of the liposome(A) comprises one or more glycolipids, such as monosialogangliosideG_(M1), or (B) is derivatized with one or more hydrophilic polymers,such as a polyethylene glycol (PEG) moiety. While not wishing to bebound by any particular theory, it is thought in the art that, at leastfor sterically stabilized liposomes containing gangliosides,sphingomyelin, or PEG-derivatized lipids, the enhanced circulationhalf-life of these sterically stabilized liposomes derives from areduced uptake into cells of the reticuloendothelial system (RES) (Allenet al., FEBS Letters, 1987, 223, 42; Wu et al., Cancer Research, 1993,53, 3765).

[0095] Various liposomes comprising one or more glycolipids are known inthe art. Papahadjopoulos et al. (Ann. N.Y. Acad. Sci., 1987, 507, 64)reported the ability of monosialoganglioside G_(M1), galactocerebrosidesulfate and phosphatidylinositol to improve blood half-lives ofliposomes. These findings were expounded upon by Gabizon et al. (Proc.Natl. Acad. Sci. U.S.A., 1988, 85, 6949). U.S. Pat. No. 4,837,028 and WO88/04924, both to Allen et al., disclose liposomes comprising (1)sphingomyelin and (2) the ganglioside G_(M1) or a galactocerebrosidesulfate ester. U.S. Pat. No. 5,543,152 (Webb et al.) discloses liposomescomprising sphingomyelin. Liposomes comprising1,2-sn-dimyristoylphosphatidylcholine are disclosed in WO 97/13499 (Limet al.).

[0096] Many liposomes comprising lipids derivatized with one or morehydrophilic polymers, and methods of preparation thereof, are known inthe art. Sunamoto et al. (Bull. Chem. Soc. Jpn., 1980, 53, 2778)described liposomes comprising a nonionic detergent, 2C₁₂15G, thatcontains a PEG moiety. Illum et al. (FEBS Lett., 1984, 167, 79) notedthat hydrophilic coating of polystyrene particles with polymeric glycolsresults in significantly enhanced blood half-lives. Syntheticphospholipids modified by the attachment of carboxylic groups ofpolyalkylene glycols (e.g., PEG) are described by Sears (U.S. Pat. Nos.4,426,330 and 4,534,899). Klibanov et al. (FEBS Lett., 1990, 268, 235)described experiments demonstrating that liposomes comprisingphosphatidylethanolamine (PE) derivatized with PEG or PEG stearate havesignificant increases in blood circulation half-lives. Blume et al.(Biochimica et Biophysica Acta, 1990, 1029, 91) extended suchobservations to other PEG-derivatized phospholipids, e.g., DSPE-PEG,formed from the combination of distearoylphosphatidylethanolamine (DSPE)and PEG. Liposomes having covalently bound PEG moieties on theirexternal surface are described in European Patent No. EP 0 445 131 B1and WO 90/04384 to Fisher. Liposome compositions containing 1-20 molepercent of PE derivatized with PEG, and methods of use thereof, aredescribed by Woodle et al. (U.S. Pat. Nos. 5,013,556 and 5,356,633) andMartin et al. (U.S. Pat. No. 5,213,804 and European Patent No. EP 0 496813 B1). Liposomes comprising a number of other lipid-polymer conjugatesare disclosed in WO 91/05545 and U.S. Pat. No. 5,225,212 (both to Martinet al.) and in WO 94/20073 (Zalipsky et al.) Liposomes comprisingPEG-modified ceramide lipids are described in WO 96/10391 (Choi et al.).U.S. Pat. Nos. 5,540,935 (Miyazaki et al.) and 5,556,948 (Tagawa et al.)describe PEG-containing liposomes that can be further derivatized withfunctional moieties on their surfaces.

[0097] A limited number of liposomes comprising nucleic acids are knownin the art. WO 96/40062 to Thierry et al. discloses methods forencapsulating high molecular weight nucleic acids in liposomes. U.S.Pat. No. 5,264,221 to Tagawa et al. discloses protein-bonded liposomesand asserts that the contents of such liposomes may include an antisenseRNA. U.S. Pat. No. 5,665,710 to Rahman et al. describes certain methodsof encapsulating oligodeoxynucleotides in liposomes. WO 97/04787 to Loveet al. discloses liposomes comprising antisense oligonucleotidestargeted to the raf gene.

[0098] Transfersomes are yet another type of liposomes, and are highlydeformable lipid aggregates which are attractive candidates for drugdelivery vehicles. Transfersomes may be described as lipid dropletswhich are so highly deformable that they are easily able to penetratethrough pores which are smaller than the droplet. Transfersomes areadaptable to the environment in which they are used, e.g. they areself-optimizing (adaptive to the shape of pores in the skin),self-repairing, frequently reach their targets without fragmenting, andoften self-loading. To make transfersomes it is possible to add surfaceedge-activators, usually surfactants, to a standard liposomalcomposition. Transfersomes have been used to deliver serum albumin tothe skin. The transfersome-mediated delivery of serum albumin has beenshown to be as effective as subcutaneous injection of a solutioncontaining serum albumin.

[0099] Surfactants find wide application in formulations such asemulsions (including microemulsions) and liposomes. The most common wayof classifying and ranking the properties of the many different types ofsurfactants, both natural and synthetic, is by the use of thehydrophile/lipophile balance (HLB). The nature of the hydrophilic group(also known as the “head”) provides the most useful means forcategorizing the different surfactants used in formulations (Rieger, inPharmaceutical Dosage Forms, Marcel Dekker, Inc., New York, N.Y., 1988,p. 285).

[0100] If the surfactant molecule is not ionized, it is classified as anonionic surfactant. Nonionic surfactants find wide application inpharmaceutical and cosmetic products and are usable over a wide range ofpH values. In general their HLB values range from 2 to about 18depending on their structure. Nonionic surfactants include nonionicesters such as ethylene glycol esters, propylene glycol esters, glycerylesters, polyglyceryl esters, sorbitan esters, sucrose esters, andethoxylated esters. Nonionic alkanolamides and ethers such as fattyalcohol ethoxylates, propoxylated alcohols, and ethoxylated/propoxylatedblock polymers are also included in this class. The polyoxyethylenesurfactants are the most popular members of the nonionic surfactantclass.

[0101] If the surfactant molecule carries a negative charge when it isdissolved or dispersed in water, the surfactant is classified asanionic. Anionic surfactants include carboxylates such as soaps, acyllactylates, acyl amides of amino acids, esters of sulfuric acid such asalkyl sulfates and ethoxylated alkyl sulfates, sulfonates such as alkylbenzene sulfonates, acyl isethionates, acyl taurates andsulfosuccinates, and phosphates. The most important members of theanionic surfactant class are the alkyl sulfates and the soaps.

[0102] If the surfactant molecule carries a positive charge when it isdissolved or dispersed in water, the surfactant is classified ascationic. Cationic surfactants include quaternary ammonium salts andethoxylated amines. The quaternary ammonium salts are the most usedmembers of this class.

[0103] If the surfactant molecule has the ability to carry either apositive or negative charge, the surfactant is classified as amphoteric.Amphoteric surfactants include acrylic acid derivatives, substitutedalkylamides, N-alkylbetaines and phosphatides.

[0104] The use of surfactants in drug products, formulations and inemulsions has been reviewed (Rieger, in Pharmaceutical Dosage Forms,Marcel Dekker, Inc., New York, N.Y., 1988, p. 285).

[0105] Penetration Enhancers

[0106] In one embodiment, the present invention employs variouspenetration enhancers to effect the efficient delivery of nucleic acids,particularly oligonucleotides, to the skin of animals. Most drugs arepresent in solution in both ionized and nonionized forms. However,usually only lipid soluble or lipophilic drugs readily cross cellmembranes. It has been discovered that even non-lipophilic drugs maycross cell membranes if the membrane to be crossed is treated with apenetration enhancer. In addition to aiding the diffusion ofnon-lipophilic drugs across cell membranes, penetration enhancers alsoenhance the permeability of lipophilic drugs.

[0107] Penetration enhancers may be classified as belonging to one offive broad categories, i.e., surfactants, fatty acids, bile salts,chelating agents, and non-chelating non-surfactants (Lee et al.,Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p.92). Eachof the above mentioned classes of penetration enhancers are describedbelow in greater detail.

[0108] Surfactants: In connection with the present invention,surfactants (or “surface-active agents”) are chemical entities which,when dissolved in an aqueous solution, reduce the surface tension of thesolution or the interfacial tension between the aqueous solution andanother liquid, with the result that absorption of oligonucleotidesthrough the mucosa is enhanced. In addition to bile salts and fattyacids, these penetration enhancers include, for example, sodium laurylsulfate, polyoxyethylene-9-lauryl ether and polyoxyethylene-20-cetylether) (Lee et al., Critical Reviews in Therapeutic Drug CarrierSystems, 1991, p.92); and perfluorochemical emulsions, such as FC-43.Takahashi et al., J. Pharm. Pharmacol., 1988, 40, 252).

[0109] Fatty acids: Various fatty acids and their derivatives which actas penetration enhancers include, for example, oleic acid, lauric acid,capric acid (n-decanoic acid), myristic acid, palmitic acid, stearicacid, linoleic acid, linolenic acid, dicaprate, tricaprate, monoolein(1-monooleoyl-rac-glycerol), dilaurin, caprylic acid, arachidonic acid,glycerol 1-monocaprate, 1-dodecylazacycloheptan-2-one, acylcarnitines,acylcholines, C₁₋₁₀ alkyl esters thereof (e.g., methyl, isopropyl andt-butyl), and mono- and di-glycerides thereof (i.e., oleate, laurate,caprate, myristate, palmitate, stearate, linoleate, etc.) (Lee et al.,Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p.92;Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems, 1990,7, 1-33; El Hariri et al., J. Pharm. Pharmacol., 1992, 44, 651-654).

[0110] Bile salts: The physiological role of bile includes thefacilitation of dispersion and absorption of lipids and fat-solublevitamins (Brunton, Chapter 38 in: Goodman & Gilman's The PharmacologicalBasis of Therapeutics, 9th Ed., Hardman et al. Eds., McGraw-Hill, NewYork, 1996, pp. 934-935). Various natural bile salts, and theirsynthetic derivatives, act as penetration enhancers. Thus the term “bilesalts” includes any of the naturally occurring components of bile aswell as any of their synthetic derivatives. The bile salts of theinvention include, for example, cholic acid (or its pharmaceuticallyacceptable sodium salt, sodium cholate), dehydrocholic acid (sodiumdehydrocholate), deoxycholic acid (sodium deoxycholate), glucholic acid(sodium glucholate), glycholic acid (sodium glycocholate),glycodeoxycholic acid (sodium glycodeoxycholate), taurocholic acid(sodium taurocholate), taurodeoxycholic acid (sodium taurodeoxycholate),chenodeoxycholic acid (sodium chenodeoxycholate), ursodeoxycholic acid(UDCA), sodium tauro-24,25-dihydro-fusidate (STDHF), sodiumglycodihydrofusidate and polyoxyethylene-9-lauryl ether (POE) (Lee etal., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, page92; Swinyard, Chapter 39 In: Remington's Pharmaceutical Sciences, 18thEd., Gennaro, ed., Mack Publishing Co., Easton, Pa., 1990, pages782-783; Muranishi, Critical Reviews in Therapeutic Drug CarrierSystems, 1990, 7, 1-33; Yamamoto et al., J. Pharm. Exp. Ther., 1992,263, 25; Yamashita et al., J. Pharm. Sci., 1990, 79, 579-583).

[0111] Chelating Agents: Chelating agents, as used in connection withthe present invention, can be defined as compounds that remove metallicions from solution by forming complexes therewith, with the result thatabsorption of oligonucleotides through the mucosa is enhanced. Withregards to their use as penetration enhancers in the present invention,chelating agents have the added advantage of also serving as DNaseinhibitors, as most characterized DNA nucleases require a divalent metalion for catalysis and are thus inhibited by chelating agents (Jarrett,J. Chromatogr., 1993, 618, 315-339). Chelating agents of the inventioninclude but are not limited to disodium ethylenediaminetetraacetate(EDTA), citric acid, salicylates (e.g., sodium salicylate,5-methoxysalicylate and homovanilate), N-acyl derivatives of collagen,laureth-9 and N-amino acyl derivatives of beta-diketones (enamines)(Leeet al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, page92; Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems,1990, 7, 1-33; Buur et al., J. Control Rel., 1990, 14, 43-51).

[0112] Non-chelating non-surfactants: As used herein, non-chelatingnon-surfactant penetration enhancing compounds can be defined ascompounds that demonstrate insignificant activity as chelating agents oras surfactants but that nonetheless enhance absorption ofoligonucleotides through the alimentary mucosa (Muranishi, CriticalReviews in Therapeutic Drug Carrier Systems, 1990, 7, 1-33). This classof penetration enhancers include, for example, unsaturated cyclic ureas,1-alkyl- and 1-alkenylazacyclo-alkanone derivatives (Lee et al.,Critical Reviews in Therapeutic Drug Carrier Systems, 1991, page 92);and non-steroidal anti-inflammatory agents such as diclofenac sodium,indomethacin and phenylbutazone (Yamashita et al., J. Pharm. Pharmacol.,1987, 39, 621-626).

[0113] Agents that enhance uptake of oligonucleotides at the cellularlevel may also be added to the pharmaceutical and other compositions ofthe present invention. For example, cationic lipids, such as lipofectin(Junichi et al, U.S. Pat. No. 5,705,188), cationic glycerol derivatives,and polycationic molecules, such as polylysine (Lollo et al., PCTApplication WO 97/30731), are also known to enhance the cellular uptakeof oligonucleotides.

[0114] Other agents may be utilized to enhance the penetration of theadministered nucleic acids, including glycols such as ethylene glycoland propylene glycol, pyrrols such as 2-pyrrol, azones, and terpenessuch as limonene and menthone.

[0115] Carriers

[0116] Certain compositions of the present invention also incorporatecarrier compounds in the formulation. As used herein, “carrier compound”or “carrier” can refer to a nucleic acid, or analog thereof, which isinert (i.e., does not possess biological activity per se) but isrecognized as a nucleic acid by in vivo processes that reduce thebioavailability of a nucleic acid having biological activity by, forexample, degrading the biologically active nucleic acid or promoting itsremoval from circulation. The coadministration of a nucleic acid and acarrier compound, typically with an excess of the latter substance, canresult in a substantial reduction of the amount of nucleic acidrecovered in the liver, kidney or other extracirculatory reservoirs,presumably due to competition between the carrier compound and thenucleic acid for a common receptor. For example, the recovery of apartially phosphorothioate oligonucleotide in hepatic tissue can bereduced when it is coadministered with polyinosinic acid, dextransulfate, polycytidic acid or4-acetamido-4′isothiocyano-stilbene-2,2′-disulfonic acid (Miyao et al.,Antisense Res. Dev., 1995, 5, 115-121; Takakura et al., Antisense &Nucl. Acid Drug Dev., 1996, 6, 177-183).

[0117] Excipients

[0118] In contrast to a carrier compound, a “pharmaceutical carrier” or“excipient” is a pharmaceutically acceptable solvent, suspending agentor any other pharmacologically inert vehicle for delivering one or morenucleic acids to an animal. The excipient may be liquid or solid and isselected, with the planned manner of administration in mind, so as toprovide for the desired bulk, consistency, etc., when combined with anucleic acid and the other components of a given pharmaceuticalcomposition. Typical pharmaceutical carriers include, but are notlimited to, binding agents (e.g., pregelatinized maize starch,polyvinylpyrrolidone or hydroxypropyl methylcellulose, etc.); fillers(e.g., lactose and other sugars, microcrystalline cellulose, pectin,gelatin, calcium sulfate, ethyl cellulose, polyacrylates or calciumhydrogen phosphate, etc.); lubricants (e.g., magnesium stearate, talc,silica, colloidal silicon dioxide, stearic acid, metallic stearates,hydrogenated vegetable oils, corn starch, polyethylene glycols, sodiumbenzoate, sodium acetate, etc.); disintegrants (e.g., starch, sodiumstarch glycolate, etc.); and wetting agents (e.g., sodium laurylsulphate, etc.).

[0119] Pharmaceutically acceptable organic or inorganic excipientsuitable for non-parenteral administration which do not deleteriouslyreact with nucleic acids can also be used to formulate the compositionsof the present invention. Suitable pharmaceutically acceptable carriersinclude, but are not limited to, water, salt solutions, alcohols,polyethylene glycols, gelatin, lactose, amylose, magnesium stearate,talc, silicic acid, viscous paraffin, hydroxymethylcellulose,polyvinylpyrrolidone and the like.

[0120] Formulations for topical administration of nucleic acids mayinclude sterile and non-sterile aqueous solutions, non-aqueous solutionsin common solvents such as alcohols, or solutions of the nucleic acidsin liquid or solid oil bases. The solutions may also contain buffers,diluents and other suitable additives. Pharmaceutically acceptableorganic or inorganic excipients suitable for non-parenteraladministration which do not deleteriously react with nucleic acids canbe used.

[0121] Suitable pharmaceutically acceptable excipients include, but arenot limited to, water, salt solutions, alcohol, polyethylene glycols,gelatin, lactose, amylose, magnesium stearate, talc, silicic acid,viscous paraffin, hydroxymethylcellulose, polyvinylpyrrolidone and thelike.

[0122] Other Components

[0123] The compositions of the present invention may additionallycontain other adjunct components conventionally found in pharmaceuticalcompositions, at their art-established usage levels. Thus, for example,the compositions may contain additional, compatible,pharmaceutically-active materials such as, for example, antipruritics,astringents, local anesthetics or anti-inflammatory agents, or maycontain additional materials useful in physically formulating variousdosage forms of the compositions of the present invention, such as dyes,flavoring agents, preservatives, antioxidants, opacifiers, thickeningagents and stabilizers. However, such materials, when added, should notunduly interfere with the biological activities of the components of thecompositions of the present invention. The formulations can besterilized and, if desired, mixed with auxiliary agents, e.g.,lubricants, preservatives, stabilizers, wetting agents, emulsifiers,salts for influencing osmotic pressure, buffers, colorings, flavoringsand/or aromatic substances and the like which do not deleteriouslyinteract with the nucleic acid(s) of the formulation.

[0124] Aqueous suspensions may contain substances which increase theviscosity of the suspension including, for example, sodiumcarboxymethylcellulose, sorbitol and/or dextran. The suspension may alsocontain stabilizers.

[0125] Certain embodiments of the invention provide pharmaceuticalcompositions containing (a) one or more antisense compounds and (b) oneor more other chemotherapeutic agents which function by a non-antisensemechanism. Examples of such chemotherapeutic agents include but are notlimited to daunorubicin, daunomycin, dactinomycin, doxorubicin,epirubicin, idarubicin, esorubicin, bleomycin, mafosfamide, ifosfamide,cytosine arabinoside, bis-chloroethylnitrosurea, busulfan, mitomycin C,actinomycin D, mithramycin, prednisone, hydroxyprogesterone,testosterone, tamoxifen, dacarbazine, procarbazine, hexamethylmelamine,pentamethylmelamine, mitoxantrone, amsacrine, chlorambucil,methylcyclohexylnitrosurea, nitrogen mustards, melphalan,cyclophosphamide, 6-mercaptopurine, 6-thioguanine, cytarabine,5-azacytidine, hydroxyurea, deoxycoformycin,4-hydroxyperoxycyclophosphoramide, 5-fluorouracil (5-FU),5-fluorodeoxyuridine (5-FUdR), methotrexate (MTX), colchicine, taxol,vincristine, vinblastine, etoposide (VP-16), trimetrexate, irinotecan,topotecan, gemcitabine, teniposide, cisplatin and diethylstilbestrol(DES). See, generally, The Merck Manual of Diagnosis and Therapy, 15thEd. 1987, pp. 1206-1228, Berkow et al., eds., Rahway, N.J. When usedwith the compounds of the invention, such chemotherapeutic agents may beused individually (e.g., 5-FU and oligonucleotide), sequentially (e.g.,5-FU and oligonucleotide for a period of time followed by MTX andoligonucleotide), or in combination with one or more other suchchemotherapeutic agents (e.g., 5-FU, MTX and oligonucleotide, or 5-FU,radiotherapy and oligonucleotide). Anti-inflammatory drugs, includingbut not limited to nonsteroidal anti-inflammatory drugs andcorticosteroids, and antiviral drugs, including but not limited toribivirin, vidarabine, acyclovir and ganciclovir, may also be combinedin compositions of the invention. See, generally, The Merck Manual-ofDiagnosis and Therapy, 15th Ed., Berkow et al., eds., 1987, Rahway,N.J., pages 2499-2506 and 46-49, respectively). Other non-antisensechemotherapeutic agents are also within the scope of this invention. Twoor more combined compounds may be used together or sequentially.

[0126] In another related embodiment, compositions of the invention maycontain one or more antisense compounds, particularly oligonucleotides,targeted to a first nucleic acid and one or more additional antisensecompounds targeted to a second nucleic acid target. Numerous examples ofantisense compounds are known in the art. Two or more combined compoundsmay be used together or sequentially.

[0127] The formulation of therapeutic compositions and their subsequentadministration is believed to be within the skill of those in the art.Dosing is dependent on severity and responsiveness of the disease stateto be treated, with the course of treatment lasting from several days toseveral months, or until a cure is effected or a diminution of thedisease state is achieved. Optimal dosing schedules can be calculatedfrom measurements of drug accumulation in the body of the patient.Persons of ordinary skill can easily determine optimum dosages, dosingmethodologies and repetition rates. Optimum dosages may vary dependingon the relative potency of individual oligonucleotides, and cangenerally be estimated based on EC₅₀s found to be effective in in vitroand in vivo animal models. In general, dosage is from 0.01 ug to 100 gper kg of body weight, and may be given once or more daily, weekly,monthly or yearly, or even once every 2 to 20 years. Persons of ordinaryskill in the art can easily estimate repetition rates for dosing basedon measured residence times and concentrations of the drug in bodilyfluids or tissues. Following successful treatment, it may be desirableto have the patient undergo maintenance therapy to prevent therecurrence of the disease state, wherein the oligonucleotide isadministered in maintenance doses, ranging from 0.01 ug to 100 g per kgof body weight, once or more daily, to once every 20 years.

[0128] While the present invention has been described with specificityin accordance with certain of its preferred embodiments, the followingexamples serve only to illustrate the invention and are not intended tolimit the same.

EXAMPLES Example 1

[0129] Nucleoside Phosphoramidites for Oligonucleotide Synthesis Deoxyand 2′-alkoxy amidites

[0130] 2′-Deoxy and 2′-methoxy beta-cyanoethyldiisopropylphosphoramidites were purchased from commercial sources (e.g. Chemgenes,Needham Mass. or Glen Research, Inc. Sterling Va.). Other 2′-O-alkoxysubstituted nucleoside amidites are prepared as described in U.S. Pat.No. 5,506,351, herein incorporated by reference. For oligonucleotidessynthesized using 2′-alkoxy amidites, optimized synthesis cycles weredeveloped that incorporate multiple steps coupling longer wait timesrelative to standard synthesis cycles.

[0131] The following abbreviations are used in the text: thin layerchromatography (TLC), melting point (MP), high pressure liquidchromatography (HPLC), Nuclear Magnetic Resonance (NMR), argon (Ar),methanol (MeOH), dichloromethane (CH₂Cl₂), triethylamine (TEA), dimethylformamide (DMF), ethyl acetate (EtOAc), dimethyl sulfoxide (DMSO),tetrahydrofuran (THF).

[0132] Oligonucleotides containing 5-methyl-2′-deoxycytidine(5-Me-dC)nucleotides were synthesized according to published methods(Sanghvi, et. al., Nucleic Acids Research, 1993, 21, 3197-3203) usingcommercially available phosphoramidites (Glen Research, Sterling Va. orChemGenes, Needham Mass.) or prepared as follows:

[0133] Preparation of 5′-O-Dimethoxytrityl-thymidine intermediate for5-methyl dC amidite

[0134] To a 50 L glass reactor equipped with air stirrer and Ar gas linewas added thymidine (1.00 kg, 4.13 mol) in anhydrous pyridine (6 L) atambient temperature. Dimethoxytrityl (DMT) chloride (1.47 kg, 4.34 mol.1.05 eq) was added as a solid in four portions over 1 h. After 30 min,TLC indicated approx. 95% product, 2% thymidine, 5% DMT reagent andby-products and 2% 3′,5′-bis DMT product (R_(f) in EtOAc 0.45, 0.05,0.98, 0.95 respectively). Saturated sodium bicarbonate (4 L) and CH₂Cl₂were added with stirring (pH of the aqueous layer 7.5). An additional 18L of water was added, the mixture was stirred, the phases wereseparated, and the organic layer was transferred to a second 50 Lvessel. The aqueous layer was extracted with additional CH₂Cl₂ (2×2 L).The combined organic layer was washed with water (10 L) and thenconcentrated in a rotary evaporator to approx. 3.6 kg total weight. Thiswas redissolved in CH₂Cl₂ (3.5 L), added to the reactor followed bywater (6 L) and hexanes (13 L). The mixture was vigorously stirred andseeded to give a fine white suspended solid starting at the interface.After stirring for 1 h, the suspension was removed by suction through a½″ diameter teflon tube into a 20 L suction flask, poured onto a 25 cmCoors Buchner funnel, washed with water (2×3 L) and a mixture ofhexanes-CH₂Cl₂ (4:1, 2×3 L) and allowed to air dry overnight in pans (1″deep). This was further dried in a vacuum oven (75° C., 0.1 mm Hg, 48 h)to a constant weight of 2072 g (93%) of a white solid, (mp 122-124° C.).TLC indicated a trace contamination of the bis DMT product. NMRspectroscopy also indicated that 1-2 mole percent pyridine and about 5mole percent of hexanes was still present.

[0135] Preparation of 51-O-Dimethoxytrityl-2′-deoxy-5-methylcytidineintermediate for 5-methyl-dC amidite

[0136] To a 50 L Schott glass-lined steel reactor equipped with anelectric stirrer, reagent addition pump (connected to an additionfunnel), heating/cooling system, internal thermometer and an Ar gas linewas added 5′-O-dimethoxytrityl-thymidine (3.00 kg, 5.51 mol), anhydrousacetonitrile (25 L) and TEA (12.3 L, 88.4 mol, 16 eq). The mixture waschilled with stirring to −10° C. internal temperature (external −20°C.). Trimethylsilylchloride (2.1 L, 16.5 mol, 3.0 eq) was added over 30minutes while maintaining the internal temperature below −5° C.,followed by a wash of anhydrous acetonitrile (1 L). Note: the reactionis mildly exothermic and copious hydrochloric acid fumes form over thecourse of the addition. The reaction was allowed to warm to 0° C. andthe reaction progress was confirmed by TLC (EtOAc-hexanes 4:1; R_(f)0.43 to 0.84 of starting material and silyl product, respectively). Uponcompletion, triazole (3.05 kg, 44 mol, 8.0 eq) was added the reactionwas cooled to −20° C. internal temperature (external −30° C.).Phosphorous oxychloride (1035 mL, 11.1 mol, 2.01 eq) was added over 60min so as to maintain the temperature between −20° C. and −10° C. duringthe strongly exothermic process, followed by a wash of anhydrousacetonitrile (1 L). The reaction was warmed to 0° C. and stirred for 1h. TLC indicated a complete conversion to the triazole product (R_(f)0.83 to 0.34 with the product spot glowing in long wavelength UV light).The reaction mixture was a peach-colored thick suspension, which turneddarker red upon warming without apparent decomposition. The reaction wascooled to −15° C. internal temperature and water (5 L) was slowly addedat a rate to maintain the temperature below +10° C. in order to quenchthe reaction and to form a homogenous solution. (Caution: this reactionis initially very strongly exothermic). Approximately one-half of thereaction volume (22 L) was transferred by air pump to another vessel,diluted with EtOAc (12 L) and extracted with water (2×8 L). The combinedwater layers were back-extracted with EtOAc (6 L). The water layer wasdiscarded and the organic layers were concentrated in a 20 L rotaryevaporator to an oily foam. The foam was coevaporated with anhydrousacetonitrile (4 L) to remove EtOAc. (note: dioxane may be used insteadof anhydrous acetonitrile if dried to a hard foam). The second half ofthe reaction was treated in the same way. Each residue was dissolved indioxane (3 L) and concentrated ammonium hydroxide (750 mL) was added. Ahomogenous solution formed in a few minutes and the reaction was allowedto stand overnight (although the reaction is complete within 1 h).

[0137] TLC indicated a complete reaction (product R_(f) 0.35 inEtOAc-MeOH 4:1). The reaction solution was concentrated on a rotaryevaporator to a dense foam. Each foam was slowly redissolved in warmEtOAc (4 L; 50° C.), combined in a 50 L glass reactor vessel, andextracted with water (2×4L) to remove the triazole by-product. The waterwas back-extracted with EtOAc (2 L). The organic layers were combinedand concentrated to about 8 kg total weight, cooled to 0° C. and seededwith crystalline product. After 24 hours, the first crop was collectedon a 25 cm Coors Buchner funnel and washed repeatedly with EtOAc (3×3L)until a white powder was left and then washed with ethyl ether (2×3L).The solid was put in pans (1″ deep) and allowed to air dry overnight.The filtrate was concentrated to an oil, then redissolved in EtOAc (2L), cooled and seeded as before. The second crop was collected andwashed as before (with proportional solvents) and the filtrate was firstextracted with water (2×1L) and then concentrated to an oil. The residuewas dissolved in EtOAc (1 L) and yielded a third crop which was treatedas above except that more washing was required to remove a yellow oilylayer.

[0138] After air-drying, the three crops were dried in a vacuum oven(50° C., 0.1 mm Hg, 24 h) to a constant weight (1750, 600 and 200 g,respectively) and combined to afford 2550 g (85%) of a white crystallineproduct (MP 215-217° C.) when TLC and NMR spectroscopy indicated purity.The mother liquor still contained mostly product (as determined by TLC)and a small amount of triazole (as determined by NMR spectroscopy), bisDMT product and unidentified minor impurities. If desired, the motherliquor can be purified by silica gel chromatography using a gradient ofMeOH (0-25%) in EtOAc to further increase the yield.

[0139] Preparation of5′-O-Dimethoxytrityl-2′-deoxy-N4-benzoyl-5-methylcytidine penultimateintermediate for 5-methyl dC amidite

[0140] Crystalline 51-O-dimethoxytrityl-5-methyl-21-deoxycytidine (2000g, 3.68 mol) was dissolved in anhydrous DMF (6.0 kg) at ambienttemperature in a 50 L glass reactor vessel equipped with an air stirrerand argon line. Benzoic anhydride (Chem Impex not Aldrich, 874 g, 3.86mol, 1.05 eq) was added and the reaction was stirred at ambienttemperature for 8 h. TLC (CH₂Cl₂-EtOAc; CH₂Cl₂-EtOAc 4:1; R_(f) 0.25)indicated approx. 92% complete reaction. An additional amount of benzoicanhydride (44 g, 0.19 mol) was added. After a total of 18 h, TLCindicated approx. 96% reaction completion. The solution was diluted withEtOAc (20 L), TEA (1020 mL, 7.36 mol, ca 2.0 eq) was added withstirring, and the mixture was extracted with water (15 L, then 2×10 L).The aqueous layer was removed (no back-extraction was needed) and theorganic layer was concentrated in 2×20 L rotary evaporator flasks untila foam began to form. The residues were coevaporated with acetonitrile(1.5 L each) and dried (0.1 mm Hg, 25° C., 24 h) to 2520 g of a densefoam. High pressure liquid chromatography (HPLC) revealed acontamination of 6.3% of N4, 3′-O-dibenzoyl product, but very littleother impurities.

[0141] THe product was purified by Biotage column chromatography (5 kgBiotage) prepared with 65:35:1 hexanes-EtOAc-TEA (4L). The crude product(800 g),dissolved in CH₂Cl₂ (2 L), was applied to the column. The columnwas washed with the 65:35:1 solvent mixture (20 kg), then 20:80:1solvent mixture (10 kg), then 99:1 EtOAc:TEA (17 kg). The fractionscontaining the product were collected, and any fractions containing theproduct and impurities were retained to be resubjected to columnchromatography. The column was reequilibrated with the original 65:35:1solvent mixture (17 kg). A second batch of crude product (840 g) wasapplied to the column as before. The column was washed with thefollowing solvent gradients: 65:35:1 (9 kg), 55:45:1 (20 kg), 20:80:1(10 kg), and 99:1 EtOAc:TEA(15 kg). The column was reequilibrated asabove, and a third batch of the crude product (850 g) plus impurefractions recycled from the two previous columns (28 g) was purifiedfollowing the procedure for the second batch. The fractions containingpure product combined and concentrated on a 20L rotary evaporator,co-evaporated with acetontirile (3 L) and dried (0.1 mm Hg, 48 h, 25°C.) to a constant weight of 2023 g (85%) of white foam and 20 g ofslightly contaminated product from the third run. HPLC indicated apurity of 99.8% with the balance as the diBenzoyl product.

[0142][5′-O-(4,4′-Dimethoxytriphenylmethyl)-2′-deoxy-N4-benzoyl-5-methylcytidin-3′-O-yl]-2-cyanoethyl-N,N-diisopropylphosphoramidite(5-methyl dC amidite)

[0143]5′-0-(4,4′-Dimethoxytriphenylmethyl)-2′-deoxy-N⁴-benzoyl-5-methylcytidine(998 g, 1.5 mol) was dissolved in anhydrous DMF (2 L). The solution wasco-evaporated with toluene (300 ml) at 50° C. under reduced pressure,then cooled to room temperature and 2-cyanoethyltetraisopropylphosphorodiamidite (680 g, 2.26 mol) and tetrazole (52.5g, 0.75 mol) were added. The mixture was shaken until all tetrazole wasdissolved, N-methylimidazole (15 ml) was added and the mixture was leftat room temperature for 5 hours. TEA (300 ml) was added, the mixture wasdiluted with DMF (2.5 L) and water (600 ml), and extracted with hexane(3×3 L). The mixture was diluted with water (1.2 L) and extracted with amixture of toluene (7.5 L) and hexane (6 L). The two layers wereseparated, the upper layer was washed with DMF-water (7:3 v/v, 3×2 L)and water (3×2 L), and the phases were separated. The organic layer wasdried (Na₂SO₄), filtered and rotary evaporated. The residue wasco-evaporated with acetonitrile (2×2 L) under reduced pressure and driedto a constant weight (25° C., 0.1 mm Hg, 40 h) to afford 1250 g anoff-white foam solid (96%).

[0144] 2′-Fluoro amidites

[0145] 21-Fluorodeoxyadenosine amidites

[0146] 2′-fluoro oligonucleotides were synthesized as describedpreviously [Kawasaki, et. al., J. Med. Chem., 1993, 36, 831-841] andU.S. Pat. No. 5,670,633, herein incorporated by reference. Thepreparation of 2′-fluoropyrimidines containing a 5-methyl substitutionare described in U.S. Pat. No. 5,861,493. Briefly, the protectednucleoside N6-benzoyl-2′-deoxy-2′-fluoroadenosine was synthesizedutilizing commercially available 9-beta-D-arabinofuranosyladenine asstarting material and whereby the 2′-alpha-fluoro atom is introduced bya S_(N)2-displacement of a 2′-beta-triflate group. ThusN6-benzoyl-9-beta-D-arabinofuranosyladenine was selectively protected inmoderate yield as the 3′,5′-ditetrahydropyranyl (THP) intermediate.Deprotection of the THP and N6-benzoyl groups was accomplished usingstandard methodologies to obtain the 5′-dimethoxytrityl-(DMT) and5′-DMT-3′-phosphoramidite intermediates.

[0147] 2′-Fluorodeoxyguanosine

[0148] The synthesis of 2′-deoxy-2′-fluoroguanosine was accomplishedusing tetraisopropyldisiloxanyl (TPDS) protected9-beta-D-arabinofuranosylguanine as starting material, and conversion tothe intermediate isobutyryl-arabinofuranosylguanosine. Alternatively,isobutyryl-arabinofuranosylguanosine was prepared as described by Rosset al., (Nucleosides & Nucleosides, 16, 1645, 1997). Deprotection of theTPDS group was followed by protection of the hydroxyl group with THP togive isobutyryl di-THP protected arabinofuranosylguanine. SelectiveO-deacylation and triflation was followed by treatment of the crudeproduct with fluoride, then deprotection of the THP groups. Standardmethodologies were used to obtain the 5′-DMT- and5′-DMT-3′-phosphoramidites.

[0149] 2′-Fluorouridine

[0150] Synthesis of 2′-deoxy-2′-fluorouridine was accomplished by themodification of a literature procedure in which2,2′-anhydro-1-beta-D-arabinofuranosyluracil was treated with 70%hydrogen fluoride-pyridine. Standard procedures were used to obtain the5′-DMT and 5′-DMT-3′phosphoramidites.

[0151] 2′-Fluorodeoxycytidine

[0152] 2′-deoxy-2′-fluorocytidine was synthesized via amination of2′-deoxy-2′-fluorouridine, followed by selective protection to giveN4-benzoyl-2′-deoxy-2′-fluorocytidine. Standard procedures were used toobtain the 5′-DMT and 5′-DMT-3′phosphoramidites.

[0153] 2′-O-(2-Methoxyethyl) modified amidites

[0154] 2′-O-Methoxyethyl-substituted nucleoside amidites (otherwiseknown as MOE amidites) are prepared as follows, or alternatively, as perthe methods of Martin, P., (Helvetica Chimica Acta, 1995, 78, 486-504).

[0155] Preparation of 2′-O-(2-methoxyethyl)-5-methyluridine intermediate

[0156] 2,2′-Anhydro-5-methyl-uridine (2000 g, 8.32 mol),tris(2-methoxyethyl)borate (2504 g, 10.60 mol), sodium bicarbonate (60g, 0.70 mol) and anhydrous 2-methoxyethanol (5 L) were combined in a 12L three necked flask and heated to 130° C. (internal temp) atatmospheric pressure, under an argon atmosphere with stirring for 21 h.TLC indicated a complete reaction. The solvent was removed under reducedpressure until a sticky gum formed (50-85° C. bath temp and 100-11 mmHg) and the residue was redissolved in water (3 L) and heated to boilingfor 30 min in order the hydrolyze the borate esters. The water wasremoved under reduced pressure until a foam began to form and then theprocess was repeated. HPLC indicated about 77% product, 15% diner (5′ ofproduct attached to 2′ of starting material) and unknown derivatives,and the balance was a single unresolved early eluting peak.

[0157] The gum was redissolved in brine (3 L), and the flask was rinsedwith additional brine (3 L). The combined aqueous solutions wereextracted with chloroform (20 L) in a heavier-than continuous extractorfor 70 h. The chloroform layer was concentrated by rotary evaporation ina 20 L flask to a sticky foam (2400 g). This was coevaporated with MeOH(400 mL) and EtOAc (8 L) at 75° C. and 0.65 atm until the foam dissolvedat which point the vacuum was lowered to about 0.5 atm. After 2.5 L ofdistillate was collected a precipitate began to form and the flask wasremoved from the rotary evaporator and stirred until the suspensionreached ambient temperature. EtOAc (2 L) was added and the slurry wasfiltered on a 25 cm table top Buchner funnel and the product was washedwith EtOAc (3×2 L). The bright white solid was air dried in pans for 24h then further dried in a vacuum oven (50° C., 0.1 mm Hg, 24 h) toafford 1649 g of a white crystalline solid (mp 115.5-116.5° C.).

[0158] The brine layer in the 20 L continuous extractor was furtherextracted for 72 h with recycled chloroform. The chloroform wasconcentrated to 120 g of oil and this was combined with the motherliquor from the above filtration (225 g), dissolved in brine (250 mL)and extracted once with chloroform (250 mL). The brine solution wascontinuously extracted and the product was crystallized as describedabove to afford an additional 178 g of crystalline product containingabout 2% of thymine. The combined yield was 1827 g (69.4%). HPLCindicated about 99.5% purity with the balance being the dimer.

[0159] Preparation of 51-O-DMT-2′-O-(2-methoxyethyl)-5-methyluridinepenultimate intermediate

[0160] In a 50 L glass-lined steel reactor,21-O-(2-methoxyethyl)-5-methyl-uridine (MOE-T, 1500 g, 4.738 mol),lutidine (1015 g, 9.476 mol) were dissolved in anhydrous acetonitrile(15 L). The solution was stirred rapidly and chilled to −10° C.(internal temperature). Dimethoxytriphenylmethyl chloride (1765.7 g,5.21 mol) was added as a solid in one portion. The reaction was allowedto warm to −2° C. over 1 h. (Note: The reaction was monitored closely byTLC (EtOAc) to determine when to stop the reaction so as to not generatethe undesired bis-DMT substituted side product). The reaction wasallowed to warm from −2 to 3° C. over 25 min. then quenched by addingMeOH (300 mL) followed after 10 min by toluene (16 L) and water (16 L).The solution was transferred to a clear 50 L vessel with a bottomoutlet, vigorously stirred for 1 minute, and the layers separated. Theaqueous layer was removed and the organic layer was washed successivelywith 10% aqueous citric acid (8 L) and water (12 L). The product wasthen extracted into the aqueous phase by washing the toluene solutionwith aqueous sodium hydroxide (0.5N, 16 L and 8 L). The combined aqueouslayer was overlayed with toluene (12 L) and solid citric acid (8 moles,1270 g) was added with vigorous stirring to lower the pH of the aqueouslayer to 5.5 and extract the product into the toluene. The organic layerwas washed with water (10 L) and TLC of the organic layer indicated atrace of DMT-O-Me, bis DMT and diner DMT.

[0161] The toluene solution was applied to a silica gel column (6 Lsintered glass funnel containing approx. 2 kg of silica gel slurriedwith toluene (2 L) and TEA(25 mL)) and the fractions were eluted withtoluene (12 L) and EtOAc (3×4 L) using vacuum applied to a filter flaskplaced below the column. The first EtOAc fraction containing both thedesired product and impurities were resubjected to column chromatographyas above. The clean fractions were combined, rotary evaporated to afoam, coevaporated with acetonitrile (6 L) and dried in a vacuum oven(0.1 mm Hg, 40 h, 40° C.) to afford 2850 g of a white crisp foam. NMRspectroscopy indicated a 0.25 mole % remainder of acetonitrile(calculates to be approx. 47 g) to give a true dry weight of 2803 g(96%). HPLC indicated that the product was 99.41% pure, with theremainder being 0.06 DMT-O-Me, 0.10 unknown, 0.44 bis DMT, and nodetectable dimer DMT or 3′-O-DMT.

[0162] Preparation of[5,-O-(4,4′-Dimethoxytriphenylmethyl)-2′-O-(2-methoxyethyl)-5-methyluridin-3′-O-yl]-2-cyanoethyl-N,N-diisopropylphosphoramidite(MOE T amidite)

[0163]5′-O-(4,4′-Dimethoxytriphenylmethyl)-2′-O-(2-methoxyethyl)-5-methyluridine(1237 g, 2.0 mol) was dissolved in anhydrous DMF (2.5 L). The solutionwas co-evaporated with toluene (200 ml) at 50° C. under reducedpressure, then cooled to room temperature and 2-cyanoethyltetraisopropylphosphorodiamidite (900 g, 3.0 mol) and tetrazole (70 g,1.0 mol) were added. The mixture was shaken until all tetrazole wasdissolved, N-methylimidazole (20 ml) was added and the solution was leftat room temperature for 5 hours. TEA (300 ml) was added, the mixture wasdiluted with DMF (3.5 L) and water (600 ml) and extracted with hexane(3×3L). The mixture was diluted with water (1.6 L) and extracted withthe mixture of toluene (12 L) and hexanes (9 L). The upper layer waswashed with DMF-water (7:3 v/v, 3×3 L) and water (3×3 L). The organiclayer was dried (Na₂SO₄), filtered and evaporated. The residue wasco-evaporated with acetonitrile (2×2 L) under reduced pressure and driedin a vacuum oven (25° C., 0.1 mm Hg, 40 h) to afford 1526 g of anoff-white foamy solid (95%).

[0164] Preparation of5′-O-Dimethoxytrityl-2′-O-(2-methoxyethyl)-5-methylcytidine intermediate

[0165] To a 50 L Schott glass-lined steel reactor equipped with anelectric stirrer, reagent addition pump (connected to an additionfunnel), heating/cooling system, internal thermometer and argon gas linewas added 5′-O-dimethoxytrityl-2′-O-(2-methoxyethyl)-5-methyl-uridine(2.616 kg, 4.23 mol, purified by base extraction only and no scrubcolumn), anhydrous acetonitrile (20 L), and TEA (9.5 L, 67.7 mol, 16eq). The mixture was chilled with stirring to −10° C. internaltemperature (external −20° C.). Trimethylsilylchloride (1.60 L, 12.7mol, 3.0 eq) was added over 30 min. while maintaining the internaltemperature below −5° C., followed by a wash of anhydrous acetonitrile(1 L). (Note: the reaction is mildly exothermic and copious hydrochloricacid fumes form over the course of the addition). The reaction wasallowed to warm to 0° C. and the reaction progress was confirmed by TLC(EtOAc, R_(f) 0.68 and 0.87 for starting material and silyl product,respectively). Upon completion, triazole (2.34 kg, 33.8 mol, 8.0 eq) wasadded the reaction was cooled to −20° C. internal temperature (external−30° C.). Phosphorous oxychloride (793 mL, 8.51 mol, 2.01 eq) was addedslowly over 60 min so as to maintain the temperature between −20° C. and−10° C. (note: strongly exothermic), followed by a wash of anhydrousacetonitrile (1 L). The reaction was warmed to 0° C. and stirred for 1h, at which point it was an off-white thick suspension. TLC indicated acomplete conversion to the triazole product (EtOAc, R_(f) 0.87 to 0.75with the product spot glowing in long wavelength UV light). The reactionwas cooled to −15° C. and water (5 L) was slowly added at a rate tomaintain the temperature below +10° C. in order to quench the reactionand to form a homogenous solution. (Caution: this reaction is initiallyvery strongly exothermic). Approximately one-half of the reaction volume(22 L) was transferred by air pump to another vessel, diluted with EtOAc(12 L) and extracted with water (2×8 L). The second half of the reactionwas treated in the same way. The combined aqueous layers wereback-extracted with EtOAc (8 L) The organic layers were combined andconcentrated in a 20 L rotary evaporator to an oily foam. The foam wascoevaporated with anhydrous acetonitrile (4 L) to remove EtOAc. (note:dioxane may be used instead of anhydrous acetonitrile if dried to a hardfoam). The residue was dissolved in dioxane (2 L) and concentratedammonium hydroxide (750 mL) was added. A homogenous solution formed in afew minutes and the reaction was allowed to stand overnight

[0166] TLC indicated a complete reaction (CH₂Cl₂-acetone-MeOH, 20:5:3,R_(f) 0. 51). The reaction solution was concentrated on a rotaryevaporator to a dense foam and slowly redissolved in warm CH₂Cl₂ (4 L,40° C.) and transferred to a 20 L glass extraction vessel equipped witha air-powered stirrer. The organic layer was extracted with water (2×6L) to remove the triazole by-product. (Note: In the first extraction anemulsion formed which took about 2 h to resolve). The water layer wasback-extracted with CH₂Cl₂ (2×2 L), which in turn was washed with water(3 L). The combined organic layer was concentrated in 2×20 L flasks to agum and then recrystallized from EtOAc seeded with crystalline product.After sitting overnight, the first crop was collected on a 25 cm CoorsBuchner funnel and washed repeatedly with EtOAc until a whitefree-flowing powder was left (about 3×3 L). The filtrate wasconcentrated to an oil recrystallized from EtOAc, and collected asabove. The solid was air-dried in pans for 48 h, then further dried in avacuum oven (50° C., 0.1 mm Hg, 17 h) to afford 2248 g of a brightwhite, dense solid (86%). An HPLC analysis indicated both crops to be99.4% pure and NMR spectroscopy indicated only a faint trace of EtOAcremained.

[0167] Preparation of5′-O-dimethoxytrityl-2′-O-(2-methoxyethyl)-N4-benzoyl-5-methyl-cytidinepenultimate intermediate:

[0168] Crystalline5′-O-dimethoxytrityl-2′-O-(2-methoxyethyl)-5-methyl-cytidine (1000 g,1.62 mol) was suspended in anhydrous DMF (3 kg) at ambient temperatureand stirred under an Ar atmosphere. Benzoic anhydride (439.3 g, 1.94mol) was added in one portion. The solution clarified after 5 hours andwas stirred for 16 h. HPLC indicated 0.45% starting material remained(as well as 0.32% N4, 3′-O-bis Benzoyl). An additional amount of benzoicanhydride (6.0 g, 0.0265 mol) was added and after 17 h, HPLC indicatedno starting material was present. TEA (450 mL, 3.24 mol) and toluene (6L) were added with stirring for 1 minute. The solution was washed withwater (4×4 L), and brine (2×4 L). The organic layer was partiallyevaporated on a 20 L rotary evaporator to remove 4 L of toluene andtraces of water. HPLC indicated that the bis benzoyl side product waspresent as a 6% impurity. The residue was diluted with toluene (7 L) andanhydrous DMSO (200 mL, 2.82 mol) and sodium hydride (60% in oil, 70 g,1.75 mol) was added in one portion with stirring at ambient temperatureover 1 h. The reaction was quenched by slowly adding then washing withaqueous citric acid (10%, 100 mL over 10 min, then 2×4 L), followed byaqueous sodium bicarbonate (2%, 2 L), water (2×4 L) and brine (4 L). Theorganic layer was concentrated on a 20 L rotary evaporator to about 2 Ltotal volume. The residue was purified by silica gel columnchromatography (6 L Buchner funnel containing 1.5 kg of silica gelwetted with a solution of EtOAc-hexanes-TEA(70:29:1)). The product waseluted with the same solvent (30 L) followed by straight EtOAc (6 L).The fractions containing the product were combined, concentrated on arotary evaporator to a foam and then dried in a vacuum oven (50° C., 0.2mm Hg, 8 h) to afford 1155 g of a crisp, white foam (98%). HPLCindicated a purity of >99.7%.

[0169] Preparation of[5′-O-(4,4′-Dimethoxytriphenylmethyl)-2′-O-(2-methoxyethyl)-N⁴-benzoyl-5-methylcytidin-3′-O-yl]-2-cyanoethyl-N,N-diisopropylphosphoramidite(MOE 5-Me-C amidite)

[0170]5′-O-(4,4′-Dimethoxytriphenylmethyl)-2-O-(2-methoxyethyl)-N⁴-benzoyl-5-methylcytidine(1082 g, 1.5 mol) was dissolved in anhydrous DMF (2 L) and co-evaporatedwith toluene (300 ml) at 50° C. under reduced pressure. The mixture wascooled to room temperature and 2-cyanoethyltetraisopropylphosphorodiamidite (680 g, 2.26 mol) and tetrazole (52.5g, 0.75 mol) were added. The mixture was shaken until all tetrazole wasdissolved, N-methylimidazole (30 ml) was added, and the mixture was leftat room temperature for 5 hours. TEA (300 ml) was added, the mixture wasdiluted with DMF (1 L) and water (400 ml) and extracted with hexane (3×3L). The mixture was diluted with water (1.2 L) and extracted with amixture of toluene (9 L) and hexanes (6 L). The two layers wereseparated and the upper layer was washed with DMF-water (60:40 v/v, 3×3L) and water (3×2 L). The organic layer was dried (Na₂SO₄), filtered andevaporated. The residue was co-evaporated with acetonitrile (2×2 L)under reduced pressure and dried in a vacuum oven (25° C., 0.1 mm Hg, 40h) to afford 1336 g of an off-white foam (97%).

[0171] Preparation of[5′-O-(4,4′-Dimethoxytriphenylmethyl)-2′-O-(2-methoxyethyl)-N⁶-benzoyladenosin-3′-O-yl]-2-cyanoethyl-N,N-diisopropylphosphoramidite(MOE A amdite)

[0172]5′-O-(4,4′-Dimethoxytriphenylmethyl)-2′-O-(2-methoxyethyl)-N⁶-benzoyladenosine(purchased from Reliable Biopharmaceutical, St. Lois, Mo.), 1098 g, 1.5mol) was dissolved in anhydrous DMF (3 L) and co-evaporated with toluene(300 ml) at 50° C. The mixture was cooled to room temperature and2-cyanoethyl tetraisopropylphosphorodiamidite (680 g, 2.26 mol) andtetrazole (78.8 g, 1.24 mol) were added. The mixture was shaken untilall tetrazole was dissolved, N-methylimidazole (30 ml) was added, andmixture was left at room temperature for 5 hours. TEA (300 ml) wasadded, the mixture was diluted with DMF (1 L) and water (400 ml) andextracted with hexanes (3×3 L). The mixture was diluted with water (1.4L) and extracted with the mixture of toluene (9 L) and hexanes (6 L).The two layers were separated and the upper layer was washed withDMF-water (60:40, v/v, 3×3 L) and water (3×2 L). The organic layer wasdried (Na₂SO₄), filtered and evaporated to a sticky foam. The residuewas co-evaporated with acetonitrile (2.5 L) under reduced pressure anddried in a vacuum oven (25° C., 0.1 mm Hg, 40 h) to afford 1350 g of anoff-white foam solid (96%).

[0173] Prepartion of[5′-O-(4,4′-Dimethoxytriphenylmethyl)-2′-O-(2-methoxyethyl)-N⁴-isobutyrylguanosin-3′-O-yl]-2-cyanoethyl-N,N-diisopropylphosphoramidite(NOE G amidite)

[0174]5′-O-(4,4′-Dimethoxytriphenylmethyl)-2′-O-(2-methoxyethyl)-N⁴-isobutyrlguanosine(purchased from Reliable Biopharmaceutical, St. Louis, Mo., 1426 g, 2.0mol) was dissolved in anhydrous DMF (2 L). The solution wasco-evaporated with toluene (200 ml) at 50° C., cooled to roomtemperature and 2-cyanoethyl tetraisopropylphosphorodiamidite (900 g,3.0 mol) and tetrazole (68 g, 0.97 mol) were added. The mixture wasshaken until all tetrazole was dissolved, N-methylimidazole (30 ml) wasadded, and the mixture was left at room temperature for 5 hours. TEA(300 ml) was added, the mixture was diluted with DMF (2 L) and water(600 ml) and extracted with hexanes (3×3 L). The mixture was dilutedwith water (2 L) and extracted with a mixture of toluene (10 L) andhexanes (5 L). The two layers were separated and the upper layer waswashed with DMF-water (60:40, v/v, 3×3 L). EtOAc (4 L) was added and thesolution was washed with water (3×4 L). The organic layer was dried(Na₂SO₄), filtered and evaporated to approx. 4 kg. Hexane (4 L) wasadded, the mixture was shaken for 10 min, and the supernatant liquid wasdecanted. The residue was co-evaporated with acetonitrile (2×2 L) underreduced pressure and dried in a vacuum oven (25° C., 0.1 mm Hg, 40 h) toafford 1660 g of an off-white foamy solid (91%).

[0175] 2′-O-(Aminooxyethyl)nucleoside amidites and2′-O-(dimethylaminooxyethyl) nucleoside amidites

[0176] 2′-(Dimethylaminooxyethoxy)nucleoside amidites2′-(Dimethylaminooxyethoxy)nucleoside amidites (also known in the art as2′-O-(dimethylaminooxyethyl)nucleoside amidites) are prepared asdescribed in the following paragraphs. Adenosine, cytidine and guanosinenucleoside amidites are prepared similarly to the thymidine(5-methyluridine) except the exocyclic amines are protected with abenzoyl moiety in the case of adenosine and cytidine and with isobutyrylin the case of guanosine.

[0177] 5′-O-tert-Butyldiphenylsilyl-O²-2′-anhydro-5-methyluridine

[0178] O²-2′-anhydro-5-methyluridine (Pro. Bio. Sint., Varese, Italy,100.0 g, 0.416 mmol), dimethylaminopyridine (0.66 g, 0.013 eq, 0.0054mmol) were dissolved in dry pyridine (500 ml) at ambient temperatureunder an argon atmosphere and with mechanical stirring.tert-Butyldiphenylchlorosilane (125.8 g, 119.0 mL, 1.1 eq, 0.458 mmol)was added in one portion. The reaction was stirred for 16 h at ambienttemperature. TLC (R_(f) 0.22, EtOAc) indicated a complete reaction. Thesolution was concentrated under reduced pressure to a thick oil. Thiswas partitioned between CH₂Cl₂ (1 L) and saturated sodium bicarbonate(2×1 L) and brine (1 L). The organic layer was dried over sodiumsulfate, filtered, and concentrated under reduced pressure to a thickoil. The oil was dissolved in a 1:1 mixture of EtOAc and ethyl ether(600 mL) and cooling the solution to −10° C. afforded a whitecrystalline solid which was collected by filtration, washed with ethylether (3×2 00 mL) and dried (40° C., 1 mm Hg, 24 h) to afford 149 g ofwhite solid (74.8%). TLC and NMR spectroscopy were consistent with pureproduct.

[0179]5′-O-tert-Butyldiphenylsilyl-2′-O-(2-hydroxyethyl)-5-methyluridine

[0180] In the fume hood, ethylene glycol (350 mL, excess) was addedcautiously with manual stirring to a 2 L stainless steel pressurereactor containing borane in tetrahydrofuran (1.0 M, 2.0 eq, 622 mL).(Caution:evolves hydrogen gas).5′-O-tert-Butyldiphenylsilyl-O²-2′-anhydro-5-methyluridine (149 g, 0.311mol) and sodium bicarbonate (0.074 g, 0.003 eq) were added with manualstirring. The reactor was sealed and heated in an oil bath until aninternal temperature of 160° C. was reached and then maintained for 16 h(pressure<100 psig). The reaction vessel was cooled to ambienttemperature and opened. TLC (EtOAc, R_(f) 0.67 for desired product andR_(f) 0.82 for ara-T side product) indicated about 70% conversion to theproduct. The solution was concentrated under reduced pressure (10 to 1mm Hg) in a warm water bath (40-100° C.) with the more extremeconditions used to remove the ethylene glycol. (Alternatively, once theTHF has evaporated the solution can be diluted with water and theproduct extracted into EtOAc). The residue was purified by columnchromatography (2 kg silica gel, EtOAc-hexanes gradient 1:1 to 4:1). Theappropriate fractions were combined, evaporated and dried to afford 84 gof a white crisp foam (50%), contaminated starting material (17.4 g, 12%recovery) and pure reusable starting material (20 g, 13% recovery). TLCand NMR spectroscopy were consistent with 99% pure product.

[0181]2′-O-([2-phthalimidoxy)ethyl]-5′-t-butyldiphenylsilyl-5-methyluridine

[0182]5′-O-tert-Butyldiphenylsilyl-2′-O-(2-hydroxyethyl)-5-methyluridine (20g, 36.98 mmol) was mixed with triphenylphosphine (11.63 g, 44.36 mmol)and N-hydroxyphthalimide (7.24g, 44.36mmol) and dried over P₂O₅ underhigh vacuum for two days at 40° C. The reaction mixture was flushed withargon and dissolved in dry THF (369.8 mL, Aldrich, sure seal bottle).Diethyl-azodicarboxylate (6.98 mL, 44.36 mmol) was added dropwise to thereaction mixture with the rate of addition maintained such that theresulting deep red coloration is just discharged before adding the nextdrop. The reaction mixture was stirred for 4 hrs., after which time TLC(EtOAc:hexane, 60:40) indicated that the reaction was complete. Thesolvent was evaporated in vacuuo and the residue purified by flashcolumn chromatography (eluted with 60:40 EtOAc:hexane), to yield2′-O-([2-phthalimidoxy)ethyl]-5′-t-butyldiphenylsilyl-5-methyluridine aswhite foam (21.819 g, 86%) upon rotary evaporation.

[0183]5′-O-tert-butyldiphenylsilyl-2′-O-[(2-formadoximinooxy)ethyl]-5-methyluridine

[0184]2′-O-([2-phthalimidoxy)ethyl]-5′-t-butyldiphenylsilyl-5-methyluridine(3.1 g, 4.5 mmol) was dissolved in dry CH₂Cl₂ (4.5 mL) andmethylhydrazine (30 mL, 4.64 mmol) was added dropwise at −10° C. to 0°C. After 1 h the mixture was filtered, the filtrate washed with ice coldCH₂Cl₂, and the combined organic phase was washed with water and brineand dried (anhydrous Na₂SO₄). The solution was filtered and evaporatedto afford 2′-O-(aminooxyethyl) thymidine, which was then dissolved inMeOH (67.5 mL). Formaldehyde (20% aqueous solution, w/w, 1.1 eq.) wasadded and the resulting mixture was stirred for 1 h. The solvent wasremoved under vacuum and the residue was purified by columnchromatography to yield5′-O-tert-butyldiphenylsilyl-2′-O-[(2-formadoximinooxy)ethyl]-5-methyluridine as white foam (1.95 g, 78%) upon rotaryevaporation.

[0185] 5′-O-tert-Butyldiphenylsilyl-2′-O-[N,Ndimethylaminooxyethyl]-5-methyluridine

[0186]5′-O-tert-butyldiphenylsilyl-2′-O-[(2-formadoximinooxy)ethyl]-5-methyluridine(1.77 g, 3.12 mmol) was dissolved in a solution of 1M pyridiniump-toluenesulfonate (PPTS) in dry MeOH (30.6 mL) and cooled to 10° C.under inert atmosphere. Sodium cyanoborohydride (0.39 g, 6.13 mmol) wasadded and the reaction mixture was stirred. After 10 minutes thereaction was warmed to room temperature and stirred for 2 h. while theprogress of the reaction was monitored by TLC (5% MeOH in CH₂Cl₂).Aqueous NaHCO₃ solution (5%, 10 mL) was added and the product wasextracted with EtOAc (2×20 mL). The organic phase was dried overanhydrous Na₂SO₄, filtered, and evaporated to dryness. This entireprocedure was repeated with the resulting residue, with the exceptionthat formaldehyde (20% w/w, 30 mL, 3.37 mol) was added upon dissolutionof the residue in the PPTS/MeOH solution. After the extraction andevaporation, the residue was purified by flash column chromatography and(eluted with 5% MeOH in CH₂Cl₂) to afford5′-O-tert-butyldiphenylsilyl-2′-O-[N,N-dimethylaminooxyethyl]-5-methyluridineas a white foam (14.6 g, 80%) upon rotary evaporation.

[0187] 2′-O-(dimethylaminooxyethyl)-5-methyluridine

[0188] Triethylamine trihydrofluoride (3.91 mL, 24.0 mmol) was dissolvedin dry THF and TEA (1.67 mL, 12 mmol, dry, stored over KOH) and added to5′-O-tert-butyldiphenylsilyl-2′-O-[N,N-dimethylaminooxyethyl]-5-methyluridine(1.40 g, 2.4 mmol). The reaction was stirred at room temperature for 24hrs and monitored by TLC (5% MeOH in CH₂Cl₂). The solvent was removedunder vacuum and the residue purified by flash column chromatography(eluted with 10% MeOH in CH₂Cl₂) to afford2′-O-(dimethylaminooxyethyl)-5-methyluridine (766 mg, 92.5%) upon rotaryevaporation of the solvent.

[0189] 5′-O-DMT-2′-O-(dimethylaminooxyethyl)-5-methyluridine

[0190] 2′-O-(dimethylaminooxyethyl)-5-methyluridine (750 mg, 2.17 mmol)was dried over P₂O₅ under high vacuum overnight at 40° C., co-evaporatedwith anhydrous pyridine (20 mL), and dissolved in pyridine (11 mL) underargon atmosphere. 4-dimethylaminopyridine (26.5 mg, 2.60 mmol) and4,4′-dimethoxytrityl chloride (880 mg, 2.60 mmol) were added to thepyridine solution and the reaction mixture was stirred at roomtemperature until all of the starting material had reacted. Pyridine wasremoved under vacuum and the residue was purified by columnchromatography (eluted with 10% MeOH in CH₂Cl₂ containing a few drops ofpyridine) to yield5′-O-DMT-2′-O-(dimethylamino-oxyethyl)-5-methyluridine (1.13 g, 80%)upon rotary evaporation.

[0191]5′-O-DMT-2′-O-(2-N,N-dimethylaminooxyethyl)-5-methyluridine-3′-[(2-cyanoethyl)-N,N-diisopropylphosphoramidite]

[0192] 5′-O-DMT-2′-O-(dimethylaminooxyethyl)-5-methyluridine (1.08 g,1.67 mmol) was co-evaporated with toluene (20 mL), N,N-diisopropylaminetetrazonide (0.29 g, 1.67 mmol) was added and the mixture was dried overP₂O₅ under high vacuum overnight at 40° C. This was dissolved inanhydrous acetonitrile (8.4 mL) and2-cyanoethyl-N,N,N¹,N¹-tetraisopropylphosphoramidite (2.12 mL, 6.08mmol) was added. The reaction mixture was stirred at ambient temperaturefor 4 h under inert atmosphere. The progress of the reaction wasmonitored by TLC (hexane:EtOAc 1:1). The solvent was evaporated, thenthe residue was dissolved in EtOAc (70 mL) and washed with 5% aqueousNaHCO₃ (40 mL). The EtOAc layer was dried over anhydrous Na₂SO₄,filtered, and concentrated. The residue obtained was purified by columnchromatography (EtOAc as eluent) to afford5′-O-DMT-2′-O-(2-N,N-dimethylaminooxyethyl)-5-methyluridine-3′-[(2-cyanoethyl)-N,N-diisopropylphosphoramidite]as a foam (1.04 g, 74.9%) upon rotary evaporation.

[0193] 2′-(Aminooxyethoxy)nucleoside amidites

[0194] 2′-(Aminooxyethoxy)nucleoside amidites (also known in the art as2¹-O-(aminooxyethyl)nucleoside amidites) are prepared as described inthe following paragraphs. Adenosine, cytidine and thymidine nucleosideamidites are prepared similarly.

[0195]N2-isobutyryl-6-O-diphenylcarbamoyl-2′-O-(2-ethylacetyl)-5′-O-(4,4′-dimethoxytrityl)guanosine-3′-[(2-cyanoethyl)-N,N-diisopropylphosphoramidite]

[0196] The 2′-O-aminooxyethyl guanosine analog may be obtained byselective 2′-O-alkylation of diaminopurine riboside. Multigramquantities of diaminopurine riboside may be purchased from Schering AG(Berlin) to provide 2′-O-(2-ethylacetyl) diaminopurine riboside alongwith a minor amount of the 3′-O-isomer.2′-O-(2-ethylacetyl)diaminopurine riboside may be resolved and convertedto 2′-O-(2-ethylacetyl)guanosine by treatment with adenosine deaminase.(McGee, D. P. C., Cook, P. D., Guinosso, C. J., WO 94/02501 A1 940203.)Standard protection procedures should afford2′-O-(2-ethylacetyl)-5′-O-(4,4′-dimethoxytrityl)guanosine and2-N-isobutyryl-6-O-diphenylcarbamoyl-2′-O-(2-ethylacetyl)-5′-O-(4,4′-dimethoxytrityl)guanosinewhich may be reduced to provide2-N-isobutyryl-6-O-diphenylcarbamoyl-2′-O-(2-hydroxyethyl)-5′-O-(4,4′-dimethoxytrityl)guanosine.As before the hydroxyl group may be displaced by N-hydroxyphthalimidevia a Mitsunobu reaction, and the protected nucleoside may bephosphitylated as usual to yield2-N-isobutyryl-6-O-diphenylcarbamoyl-2′-O-([2-phthalmidoxy]ethyl)-5′-O-(4,4′-dimethoxytrityl)guanosine-3′-[(2-cyanoethyl)-N,N-diisopropylphosphoramidite].

[0197] 2′-dimethylaminoethoxyethoxy (2′-DHAEOE)nucleoside amidites

[0198] 2′-dimethylaminoethoxyethoxy nucleoside amidites (also known inthe art as 2′-O-dimethylaminoethoxyethyl, i.e., 2′-O—CH₂—O—CH₂—N(CH₂)₂,or 2′-DMAEOE nucleoside amidites) are prepared as follows. Othernucleoside amidites are prepared similarly.

[0199] 2′-O-[2(2-N,N-dimethylaminoethoxy)ethyl]-5-methyl uridine

[0200] 2[2-(Dimethylamino)ethoxy]ethanol (Aldrich, 6.66 g, 50 mmol) wasslowly added to a solution of borane in tetra-hydrofuran (1 M, 10 mL, 10mmol) with stirring in a 100 mL bomb. (Caution: Hydrogen gas evolves asthe solid dissolves). O²—,2′-anhydro-5-methyluridine (1.2 g, 5 mmol),and sodium bicarbonate (2.5 mg) were added and the bomb was sealed,placed in an oil bath and heated to 155° C. for 26 h. then cooled toroom temperature. The crude solution was concentrated, the residue wasdiluted with water (200 mL) and extracted with hexanes (200 mL). Theproduct was extracted from the aqueous layer with EtOAc (3×200 mL) andthe combined organic layers were washed once with water, dried overanhydrous sodium sulfate, filtered and concentrated. The residue waspurified by silica gel column chromatography (eluted with 5:100:2MeOH/CH₂Cl₂/TEA) as the eluent. The appropriate fractions were combinedand evaporated to afford the product as a white solid.

[0201]5′-O-dimethoxytrityl-2′-O-[2(2-N,N-dimethylaminoethoxy)ethyl)]-5-methyluridine

[0202] To 0.5 g (1.3 mmol) of2′-O-[2(2-N,N-dimethylamino-ethoxy)ethyl)]-5-methyl uridine in anhydrouspyridine (8 mL), was added TEA (0.36 mL) and dimethoxytrityl chloride(DMT-Cl, 0.87 g, 2 eq.) and the reaction was stirred for 1 h. Thereaction mixture was poured into water (200 mL) and extracted withCH₂Cl₂ (2×200 mL). The combined CH₂Cl₂ layers were washed with saturatedNaHCO₃ solution, followed by saturated NaCl solution, dried overanhydrous sodium sulfate, filtered and evaporated. The residue waspurified by silica gel column chromatography (eluted with 5:100:1MeOH/CH₂Cl₂/TEA) to afford the product.

[0203]5′-O-Dimethoxytrityl-2′-O-[2(2-N,N-dimethylaminoethoxy)-ethyl)]-5-methyluridine-3′-O-(cyanoethyl-N,N-diisopropyl)phosphoramidite

[0204] Diisopropylaminotetrazolide (0.6 g) and2-cyanoethoxy-N,N-diisopropyl phosphoramidite (1.1 mL, 2 eq.) were addedto a solution of5′-O-dimethoxytrityl-2′-O-[2(2-N,N-dimethylaminoethoxy)ethyl)]-5-methyluridine(2.17 g, 3 mmol) dissolved in CH₂Cl₂ (20 mL) under an atmosphere ofargon. The reaction mixture was stirred overnight and the solventevaporated. The resulting residue was purified by silica gel columnchromatography with EtOAc as the eluent to afford the title compound.

Example 2

[0205] Oligonucleotide Synthesis

[0206] Unsubstituted and substituted phosphodiester (P═O)oligonucleotides are synthesized on an automated DNA synthesizer(Applied Biosystems model 394) using standard phosphoramidite chemistrywith oxidation by iodine.

[0207] Phosphorothioates (P═S) are synthesized similar to phosphodiesteroligonucleotides with the following exceptions: thiation was effected byutilizing a 10% w/v solution of 3H-1,2-benzodithiole-3-one 1,1-dioxidein acetonitrile for the oxidation of the phosphite linkages. Thethiation reaction step time was increased to 180 sec and preceded by thenormal capping step. After cleavage from the CPG column and deblockingin concentrated ammonium hydroxide at 55° C. (12-16 hr), theoligonucleotides were recovered by precipitating with >3 volumes ofethanol from a 1 M NH₄oAc solution. Phosphinate oligonucleotides areprepared as described in U.S. Pat. No. 5,508,270, herein incorporated byreference.

[0208] Alkyl phosphonate oligonucleotides are prepared as described inU.S. Pat. No. 4,469,863, herein incorporated by reference.

[0209] 3′-Deoxy-3′-methylene phosphonate oligonucleotides are preparedas described in U.S. Pat. Nos. 5,610,289 or 5,625,050, hereinincorporated by reference.

[0210] Phosphoramidite oligonucleotides are prepared as described inU.S. Pat. No., 5,256,775 or U.S. Pat. No. 5,366,878, herein incorporatedby reference.

[0211] Alkylphosphonothioate oligonucleotides are prepared as describedin published PCT applications PCT/US94/00902 and PCT/US93/06976(published as WO 94/17093 and WO 94/02499, respectively), hereinincorporated by reference.

[0212] 3′-Deoxy-3′-amino phosphoramidate oligonucleotides are preparedas described in U.S. Pat. No. 5,476,925, herein incorporated byreference.

[0213] Phosphotriester oligonucleotides are prepared as described inU.S. Pat. No. 5,023,243, herein incorporated by reference.

[0214] Borano phosphate oligonucleotides are prepared as described inU.S. Pat. Nos. 5,130,302 and 5,177,198, both herein incorporated byreference.

Example 3

[0215] Oligonucleoside Synthesis

[0216] Methylenemethylimino linked oligonucleosides, also identified asMMI linked oligonucleosides, methylenedimethyl-hydrazo linkedoligonucleosides, also identified as MDH linked oligonucleosides, andmethylenecarbonylamino linked oligonucleosides, also identified asamide-3 linked oligonucleosides, and methyleneaminocarbonyl linkedoligonucleosides, also identified as amide-4 linked oligonucleosides, aswell as mixed backbone compounds having, for instance, alternating MMIand P⊚O or P═S linkages are prepared as described in U.S. Pat. Nos.5,378,825, 5,386,023, 5,489,677, 5,602,240 and 5,610,289, all of whichare herein incorporated by reference.

[0217] Formacetal and thioformacetal linked oligonucleosides areprepared as described in U.S. Pat. Nos 5,264,562 and 5,264,564, hereinincorporated by reference.

[0218] Ethylene oxide linked oligonucleosides are prepared as describedin U.S. Pat. No. 5,223,618, herein incorporated by reference.

Example 4

[0219] PNA Synthesis

[0220] Peptide nucleic acids (PNAs) are prepared in accordance with anyof the various procedures referred to in Peptide Nucleic Acids (PNA):Synthesis, Properties and Potential Applications, Bioorganic & MedicinalChemistry, 1996, 4, 5-23. They may also be prepared in accordance withU.S. Pat. Nos. 5,539,082, 5,700,922, and 5,719,262, herein incorporatedby reference.

Example 5

[0221] Synthesis of Chimeric Oligonucleotides

[0222] Chimeric oligonucleotides, oligonucleosides or mixedoligonucleotides/oligonucleosides of the invention can be of severaldifferent types. These include a first type wherein the “gap” segment oflinked nucleosides is positioned between 5′ and 3′ “wing” segments oflinked nucleosides and a second “open end” type wherein the “gap”segment is located at either the 3′ or the 5′ terminus of the oligomericcompound. Oligonucleotides of the first type are also known in the artas “gapmers” or gapped oligonucleotides. Oligonucleotides of the secondtype are also known in the art as “hemimers” or “wingmers”.

[0223] [2′-O—Me]-[2′-deoxy]-[2′-O—Me]Chimeric PhosphorothioateOligonucleotides

[0224] Chimeric oligonucleotides having 2′-O-alkyl phosphorothioate and2′-deoxy phosphorothioate oligonucleotide segments are synthesized usingan Applied Biosystems automated DNA synthesizer Model 394, as above.Oligonucleotides are synthesized using the automated synthesizer and2′-deoxy-5′-dimethoxytrityl-3′-O-phosphor-amidite for the DNA portionand 5′-dimethoxytrityl-2′-O-methyl-3′-O-phosphoramidite for 5′ and 3′wings. The standard synthesis cycle is modified by incorporatingcoupling steps with increased reaction times for the5′-dimethoxytrityl-2′-O-methyl-3′-O-phosphoramidite. The fully protectedoligonucleotide is cleaved from the support and deprotected inconcentrated ammonia (NH₄OH) for 12-16 hr at 55° C. The deprotectedoligo is then recovered by an appropriate method (precipitation, columnchromatography, volume reduced in vacuo and analyzedspetrophotometrically for yield and for purity by capillaryelectrophoresis and by mass spectrometry.

[0225] [2′-O-(2-Methoxyethyl)]-[2′-deoxy]-[2′-O-(Methoxyethyl)]ChimericPhosphorothioate Oligonucleotides

[0226] [2′-O-(2-methoxyethyl)]-[2′-deoxy]-[-2′-O-(methoxyethyl)]chimericphosphorothioate oligonucleotides were prepared as per the procedureabove for the 2′-O-methyl chimeric oligonucleotide, with thesubstitution of 2′-O-(methoxyethyl)amidites for the 2′-O-methylamidites.

[0227] [2′-O-(2-Methoxyethyl)Phosphodiester]-[2′-deoxyPhosphorothioate]-[2′-O-(2-Methoxyethyl)Phosphodiester]ChimericOligonucleotides

[0228] [2′-O-(2-methoxyethyl phosphodiester]-[2′-deoxyphosphorothioate]-[2′-O-(methoxyethyl)phosphodiester]chimericoligonucleotides are prepared as per the above procedure for the2′-O-methyl chimeric oligonucleotide with the substitution of2′-O-(methoxyethyl)amidites for the 2′-O-methyl amidites, oxidation withiodine to generate the phosphodiester internucleotide linkages withinthe wing portions of the chimeric structures and sulfurization utilizing3,H-1,2 benzodithiole-3-one 1,1 dioxide (Beaucage Reagent) to generatethe phosphorothioate internucleotide linkages for the center gap.

[0229] Other chimeric oligonucleotides, chimeric oligonucleosides andmixed chimeric oligonucleotides/oligonucleosides are synthesizedaccording to U.S. Pat. No. 5,623,065, herein incorporated by reference.

Example 6

[0230] Oligonucleotide Isolation

[0231] After cleavage from the controlled pore glass solid support anddeblocking in concentrated ammonium hydroxide at 55° C. for 12-16 hours,the oligonucleotides or oligonucleosides are recovered by precipitationout of 1 M NH₄OAc with >3 volumes of ethanol. Synthesizedoligonucleotides were analyzed by electrospray mass spectroscopy(molecular weight determination) and by capillary gel electrophoresisand judged to be at least 70% full length material. The relative amountsof phosphorothioate and phosphodiester linkages obtained in thesynthesis was determined by the ratio of correct molecular weightrelative to the −16 amu product (±32 ±48). For some studiesoligonucleotides were purified by HPLC, as described by Chiang et al.,J. Biol. Chem. 1991, 266, 18162-18171. Results obtained withHPLC-purified material were similar to those obtained with non-HPLCpurified material.

Example 7

[0232] Oligonucleotide Synthesis—96 Well Plate Format

[0233] Oligonucleotides were synthesized via solid phase P(III)phosphoramidite chemistry on an automated synthesizer capable ofassembling 96 sequences simultaneously in a 96-well format.Phosphodiester internucleotide linkages were afforded by oxidation withaqueous iodine. Phosphorothioate internucleotide linkages were generatedby sulfurization utilizing 3,H-1,2 benzodithiole-3-one 1,1 dioxide(Beaucage Reagent) in anhydrous acetonitrile. Standard base-protectedbeta-cyanoethyl-diiso-propyl phosphoramidites were purchased fromcommercial vendors (e.g. PE-Applied Biosystems, Foster City, Calif., orPharmacia, Piscataway, N.J.). Non-standard nucleosides are synthesizedas per standard or patented methods. They are utilized as base protectedbeta-cyanoethyldiisopropyl phosphoramidites.

[0234] Oligonucleotides were cleaved from support and deprotected withconcentrated NH₄OH at elevated temperature (55-60° C.) for 12-16 hoursand the released product then dried in vacuo. The dried product was thenre-suspended in sterile water to afford a master plate from which allanalytical and test plate samples are then diluted utilizing roboticpipettors.

Example 8

[0235] Oligonucleotide Analysis—96-Well Plate Format

[0236] The concentration of oligonucleotide in each well was assessed bydilution of samples and UV absorption spectroscopy. The full-lengthintegrity of the individual products was evaluated by capillaryelectrophoresis (CE) in either the 96-well format (Beckman P/ACE™ MDQ)or, for individually prepared samples, on a commercial CE apparatus(e.g., Beckman P/ACE™ 5000, ABI 270). Base and backbone composition wasconfirmed by mass analysis of the compounds utilizing electrospray-massspectroscopy. All assay test plates were diluted from the master plateusing single and multi-channel robotic pipettors. Plates were judged tobe acceptable if at least 85% of the compounds on the plate were atleast 85% full length.

Example 9

[0237] Cell Culture and Oligonucleotide Treatment

[0238] The effect of antisense compounds on target nucleic acidexpression can be tested in any of a variety of cell types provided thatthe target nucleic acid is present at measurable levels. This can beroutinely determined using, for example, PCR or Northern blot analysis.The following cell types are provided for illustrative purposes, butother cell types can be routinely used, provided that the target isexpressed in the cell type chosen. This can be readily determined bymethods routine in the art, for example Northern blot analysis,ribonuclease protection assays, or RT-PCR.

[0239] T-24 Cells:

[0240] The human transitional cell bladder carcinoma cell line T-24 wasobtained from the American Type Culture Collection (ATCC) (Manassas,Va.). T-24 cells were routinely cultured in complete McCoy's 5A basalmedia (Invitrogen Corporation, Carlsbad, Calif.) supplemented with 10%fetal calf serum (Invitrogen Corporation, Carlsbad, Calif.), penicillin100 units per mL, and streptomycin 100 micrograms per mL (InvitrogenCorporation, Carlsbad, Calif.). Cells were routinely passaged bytrypsinization and dilution when they reached 90% confluence. Cells wereseeded into 96-well plates (Falcon-Primaria #3872) at a density of 7000cells/well for use in RT-PCR analysis.

[0241] For Northern blotting or other analysis, cells may be seeded onto100 mm or other standard tissue culture plates and treated similarly,using appropriate volumes of medium and oligonucleotide.

[0242] A549 Cells:

[0243] The human lung carcinoma cell line A549 was obtained from theAmerican Type Culture Collection (ATCC) (Manassas, Va.). A549 cells wereroutinely cultured in DMEM basal media (Invitrogen Corporation,Carlsbad, Calif.) supplemented with 10% fetal calf serum (InvitrogenCorporation, Carlsbad, Calif.), penicillin 100 units per mL, andstreptomycin 100 micrograms per mL (Invitrogen Corporation, Carlsbad,Calif.). Cells were routinely passaged by trypsinization and dilutionwhen they reached 90% confluence.

[0244] NHDF Cells:

[0245] Human neonatal dermal fibroblast (NHDF) were obtained from theClonetics Corporation (Walkersville, Md.). NHDFs were routinelymaintained in Fibroblast Growth Medium (Clonetics Corporation,Walkersville, Md.) supplemented as recommended by the supplier. Cellswere maintained for up to 10 passages as recommended by the supplier.

[0246] HEK Cells:

[0247] Human embryonic keratinocytes (HEK) were obtained from theClonetics Corporation (Walkersville, Md.). HEKs were routinelymaintained in Keratinocyte Growth Medium (Clonetics Corporation,Walkersville, Md.) formulated as recommended by the supplier. Cells wereroutinely maintained for up to 10 passages as recommended by thesupplier.

[0248] Treatment with Antisense Compounds:

[0249] When cells reached 70% confluency, they were treated witholigonucleotide. For cells grown in 96-well plates, wells were washedonce with 100 μL OPTI-MEM™-1 reduced-serum medium (InvitrogenCorporation, Carlsbad, Calif.) and then treated with 130 μL ofOPTI-MEM™-1 containing 3.75 μg/mL LIPOFECTIN™ (Invitrogen Corporation,Carlsbad, Calif.) and the desired concentration of oligonucleotide.After 4-7 hours of treatment, the medium was replaced with fresh medium.Cells were harvested 16-24 hours after oligonucleotide treatment.

[0250] The concentration of oligonucleotide used varies from cell lineto cell line. To determine the optimal oligonucleotide concentration fora particular cell line, the cells are treated with a positive controloligonucleotide at a range of concentrations. For human cells thepositive control oligonucleotide is selected from either ISIS 13920(TCCGTCATCGCTCCTCAGGG, SEQ ID NO: 1) which is targeted to human H-ras,or ISIS 18078, (GTGCGCGCGAGCCCGAAATC, SEQ ID NO: 2) which is targeted tohuman Jun-N-terminal kinase-2 (JNK2). Both controls are2′-O-methoxyethyl gapmers (2′-O-methoxyethyls shown in bold) with aphosphorothioate backbone. For mouse or rat cells the positive controloligonucleotide is ISIS 15770, ATGCATTCTGCCCCCAAGGA, SEQ ID NO: 3, a2′-O-methoxyethyl gapmer (2′-O-methoxyethyls shown in bold) with aphosphorothioate backbone which is targeted to both mouse and rat c-raf.The concentration of positive control oligonucleotide that results in80% inhibition of c-Ha-ras (for ISIS 13920) or c-raf (for ISIS 15770)mRNA is then utilized as the screening concentration for newoligonucleotides in subsequent experiments for that cell line. If 80%inhibition is not achieved, the lowest concentration of positive controloligonucleotide that results in 60% inhibition of H-ras or c-raf mRNA isthen utilized as the oligonucleotide screening concentration insubsequent experiments for that cell line. If 60% inhibition is notachieved, that particular cell line is deemed as unsuitable foroligonucleotide transfection experiments. The concentrations ofantisense oligonucleotides used herein are from 50 nM to 300 nM.

Example 10

[0251] Analysis of Oligonucleotide Inhibition of Hypothetical TumorEndothelial Marker Expression

[0252] Antisense modulation of hypothetical tumor endothelial markerexpression can be assayed in a variety of ways known in the art. Forexample, hypothetical tumor endothelial marker mRNA levels can bequantitated by, e.g., Northern blot analysis, competitive polymerasechain reaction (PCR), or real-time PCR (RT-PCR). Real-time quantitativePCR is presently preferred. RNA analysis can be performed on totalcellular RNA or poly(A)+ mRNA. The preferred method of RNA analysis ofthe present invention is the use of total cellular RNA as described inother examples herein. Methods of RNA isolation are taught in, forexample, Ausubel, F. M. et al., Current Protocols in Molecular Biology,Volume 1, pp. 4.1.1-4.2.9 and 4.5.1-4.5.3, John Wiley & Sons, Inc.,1993. Northern blot analysis is routine in the art and is taught in, forexample, Ausubel, F. M. et al., Current Protocols in Molecular Biology,Volume 1, pp. 4.2.1-4.2.9, John Wiley & Sons, Inc., 1996. Real-timequantitative (PCR) can be conveniently accomplished using thecommercially available ABI PRISM™ 7700 Sequence Detection System,available from PE-Applied Biosystems, Foster City, Calif. and usedaccording to manufacturer's instructions.

[0253] Protein levels of hypothetical tumor endothelial marker can bequantitated in a variety of ways well known in the art, such asimmunoprecipitation, Western blot analysis (immunoblotting), ELISA orfluorescence-activated cell sorting (FACS). Antibodies directed tohypothetical tumor endothelial marker can be identified and obtainedfrom a variety of sources, such as the MSRS catalog of antibodies (AerieCorporation, Birmingham, Mich.), or can be prepared via conventionalantibody generation methods. Methods for preparation of polyclonalantisera are taught in, for example, Ausubel, F. M. et al., (CurrentProtocols in Molecular Biology, Volume 2, pp. 11.12.1-11.12.9, JohnWiley & Sons, Inc., 1997). Preparation of monoclonal antibodies istaught in, for example, Ausubel, F. M. et al., (Current Protocols inMolecular Biology, Volume 2, pp. 11.4.1-11.11.5, John Wiley & Sons,Inc., 1997).

[0254] Immunoprecipitation methods are standard in the art and can befound at, for example, Ausubel, F. M. et al., (Current Protocols inMolecular Biology, Volume 2, pp. 10.16.1-10.16.11, John Wiley & Sons,Inc., 1998). Western blot (immunoblot) analysis is standard in the artand can be found at, for example, Ausubel, F. M. et al., (CurrentProtocols in Molecular Biology, Volume 2, pp. 10.8.1-10.8.21, John Wiley& Sons, Inc., 1997). Enzyme-linked immunosorbent assays (ELISA) arestandard in the art and can be found at, for example, Ausubel, F. M. etal., (Current Protocols in Molecular Biology, Volume 2, pp.11.2.1-11.2.22, John Wiley & Sons, Inc., 1991).

Example 11

[0255] Poly(A)+ mRNA Isolation

[0256] Poly(A)+ mRNA was isolated according to Miura et al., (Clin.Chem., 1996, 42, 1758-1764). Other methods for poly(A)+ mRNA isolationare taught in, for example, Ausubel, F. M. et al., (Current Protocols inMolecular Biology, Volume 1, pp. 4.5.1-4.5.3, John Wiley & Sons, Inc.,1993). Briefly, for cells grown on 96-well plates, growth medium wasremoved from the cells and each well was washed with 200 μL cold PBS. 60μL lysis buffer (10 mM Tris-HCl, pH 7.6, 1 mM EDTA, 0.5 M NaCl, 0.5%NP-40, 20 mM vanadyl-ribonucleoside complex) was added to each well, theplate was gently agitated and then incubated at room temperature forfive minutes. 55 μL of lysate was transferred to Oligo d(T) coated96-well plates (AGCT Inc., Irvine Calif.). Plates were incubated for 60minutes at room temperature, washed 3 times with 200 μL of wash buffer(10 mM Tris-HCl pH 7.6, 1 mM EDTA, 0.3 M NaCl). After the final wash,the plate was blotted on paper towels to remove excess wash buffer andthen air-dried for 5 minutes. 60 μL of elution buffer (5 mM Tris-HCl pH7.6), preheated to 70° C., was added to each well, the plate wasincubated on a 90° C. hot plate for 5 minutes, and the eluate was thentransferred to a fresh 96-well plate.

[0257] Cells grown on 100 mm or other standard plates may be treatedsimilarly, using appropriate volumes of all solutions.

Example 12

[0258] Total RNA Isolation

[0259] Total RNA was isolated using an RNEASY 96™ kit and bufferspurchased from Qiagen Inc. (Valencia, Calif.) following themanufacturer's recommended procedures. Briefly, for cells grown on96-well plates, growth medium was removed from the cells and each wellwas washed with 200 μL cold PBS. 150 μL Buffer RLT was added to eachwell and the plate vigorously agitated for 20 seconds. 150 μL of 70%ethanol was then added to each well and the contents mixed by pipettingthree times up and down. The samples were then transferred to the RNEASY96™ well plate attached to a QIAVAC™ manifold fitted with a wastecollection tray and attached to a vacuum source. Vacuum was applied for1 minute. 500 μL of Buffer RW1 was added to each well of the RNEASY 96™plate and incubated for 15 minutes and the vacuum was again applied for1 minute. An additional 500 μL of Buffer RW1 was added to each well ofthe RNEASY 96™ plate and the vacuum was applied for 2 minutes. 1 mL ofBuffer RPE was then added to each well of the RNEASY 96™ plate and thevacuum applied for a period of 90 seconds. The Buffer RPE wash was thenrepeated and the vacuum was applied for an additional 3 minutes. Theplate was then removed from the QIAVAC™ manifold and blotted dry onpaper towels. The plate was then re-attached to the QIAVAC™ manifoldfitted with a collection tube rack containing 1.2 mL collection tubes.RNA was then eluted by pipetting 170 μL water into each well, incubating1 minute, and then applying the vacuum for 3 minutes.

[0260] The repetitive pipetting and elution steps may be automated usinga QIAGEN Bio-Robot 9604 (Qiagen, Inc., Valencia Calif.). Essentially,after lysing of the cells on the culture plate, the plate is transferredto the robot deck where the pipetting, DNase treatment and elution stepsare carried out.

Example 13

[0261] Real-time Quantitative PCR Analysis of Hypothetical TumorEndothelial Marker mRNA Levels

[0262] Quantitation of hypothetical tumor endothelial marker mRNA levelswas determined by real-time quantitative PCR using the ABI PRISM™ 7700Sequence Detection System (PE-Applied Biosystems, Foster City, Calif.)according to manufacturer's instructions. This is a closed-tube,non-gel-based, fluorescence detection system which allowshigh-throughput quantitation of polymerase chain reaction (PCR) productsin real-time. As opposed to standard PCR in which amplification productsare quantitated after the PCR is completed, products in real-timequantitative PCR are quantitated as they accumulate. This isaccomplished by including in the PCR reaction an oligonucleotide probethat anneals specifically between the forward and reverse PCR primers,and contains two fluorescent dyes. A reporter dye (e.g., FAM or JOE,obtained from either PE-Applied Biosystems, Foster City, Calif., OperonTechnologies Inc., Alameda, Calif. or Integrated DNA Technologies Inc.,Coralville, Iowa) is attached to the 5′ end of the probe and a quencherdye (e.g., TAMRA, obtained from either PE-Applied Biosystems, FosterCity, Calif., Operon Technologies Inc., Alameda, Calif. or IntegratedDNA Technologies Inc., Coralville, Iowa) is attached to the 3′ end ofthe probe. When the probe and dyes are intact, reporter dye emission isquenched by the proximity of the 3′ quencher dye. During amplification,annealing of the probe to the target sequence creates a substrate thatcan be cleaved by the 5′-exonuclease activity of Taq polymerase. Duringthe extension phase of the PCR amplification cycle, cleavage of theprobe by Taq polymerase releases the reporter dye from the remainder ofthe probe (and hence from the quencher moiety) and a sequence-specificfluorescent signal is generated. With each cycle, additional reporterdye molecules are cleaved from their respective probes, and thefluorescence intensity is monitored at regular intervals by laser opticsbuilt into the ABI PRISMS 7700 Sequence Detection System. In each assay,a series of parallel reactions containing serial dilutions of mRNA fromuntreated control samples generates a standard curve that is used toquantitate the percent inhibition after antisense oligonucleotidetreatment of test samples.

[0263] Prior to quantitative PCR analysis, primer-probe sets specific tothe target gene being measured are evaluated for their ability to be“multiplexed” with a GAPDH amplification reaction. In multiplexing, boththe target gene and the internal standard gene GAPDH are amplifiedconcurrently in a single sample. In this analysis, mRNA isolated fromuntreated cells is serially diluted. Each dilution is amplified in thepresence of primer-probe sets specific for GAPDH only, target gene only(“single-plexing”), or both (multiplexing). Following PCR amplification,standard curves of GAPDH and target mRNA signal as a function ofdilution are generated from both the single-plexed and multiplexedsamples. If both the slope and correlation coefficient of the GAPDH andtarget signals generated from the multiplexed samples fall within 10% oftheir corresponding values generated from the single-plexed samples, theprimer-probe set specific for that target is deemed multiplexable. Othermethods of PCR are also known in the art.

[0264] PCR reagents were obtained from Invitrogen Corporation,(Carlsbad, Calif.). RT-PCR reactions were carried out by adding 20 μLPCR cocktail (2.5×PCR buffer (—MgCl₂), 6.6 mM MgCl₂, 375 , each of DATP,dCTP, dCTP and dGTP, 375 nM each of forward primer and reverse primer,125 nM of probe, 4 Units RNAse inhibitor, 1.25 Units PLATINUM® Taq, 5Units MuLV reverse transcriptase, and 2.5×ROX dye) to 96-well platescontaining 30 μL total RNA solution. The RT reaction was carried out byincubation for 30 minutes at 48° C. Following a 10 minute incubation at95° C. to activate the PLATINUM® Taq, 40 cycles of a two-step PCRprotocol were carried out: 95° C. for 15 seconds (denaturation) followedby 60° C. for 1.5 minutes (annealing/extension).

[0265] Gene target quantities obtained by real time RT-PCR arenormalized using either the expression level of GAPDH, a gene whoseexpression is constant, or by quantifying total RNA using RiboGreen™(Molecular Probes, Inc. Eugene, Or.). GAPDH expression is quantified byreal time RT-PCR, by being run simultaneously with the target,multiplexing, or separately. Total RNA is quantified using RiboGreen™RNA quantification reagent from Molecular Probes. Methods of RNAquantification by RiboGreen™ are taught in Jones, L. J., et al,(Analytical Biochemistry, 1998, 265, 368-374).

[0266] In this assay, 170 μL of RiboGreen™ working reagent (RiboGreen™reagent diluted 1:350 in 10 mM Tris-HCl, 1 mM EDTA, pH 7.5) is pipettedinto a 96-well plate containing 30 μL purified, cellular RNA. The plateis read in a CytoFluor 4000 (PE Applied Biosystems) with excitation at480 nm and emission at 520 nm.

[0267] Probes and primers to human hypothetical tumor endothelial markerwere designed to hybridize to a human hypothetical tumor endothelialmarker sequence, using published sequence information (GenBank accessionnumber AB062750.1, incorporated herein as SEQ ID NO: 4). For humanhypothetical tumor endothelial marker the PCR primers were: forwardprimer: CTGGGCCACATCAGAATAAGG (SEQ ID NO: 5) reverse primer:CGCAATCAGGACAGTCAGCAT (SEQ ID NO: 6) and the PCR probe was:FAM-CTTGGCCCCATCCTCTCACAGCCTAG-TAMRA (SEQ ID NO: 7) where FAM is thefluorescent dye and TAMRA is the quencher dye. For human GAPDH the PCRprimers were: forward primer: GAAGGTGAAGGTCGGAGTC(SEQ ID NO: 8) reverseprimer: GAAGATGGTGATGGGATTTC (SEQ ID NO: 9) and the PCR probe was: 5′JOE-CAAGCTTCCCGTTCTCAGCC-TAMRA 3′ (SEQ ID NO: 10) where JOE is thefluorescent reporter dye and TAMRA is the quencher dye.

Example 14

[0268] Northern Blot Analysis of Hypothetical Tumor Endothelial MarkermRNA Levels

[0269] Eighteen hours after antisense treatment, cell monolayers werewashed twice with cold PBS and lysed in 1 mL RNAZOL™ (TEL-TEST “B” Inc.,Friendswood, Tex.). Total RNA was prepared following manufacturer'srecommended protocols. Twenty micrograms of total RNA was fractionatedby electrophoresis through 1.2% agarose gels containing 1.1%formaldehyde using a MOPS buffer system (AMRESCO, Inc. Solon, Ohio). RNAwas transferred from the gel to HYBOND™-N+ nylon membranes (AmershamPharmacia Biotech, Piscataway, N.J.) by overnight capillary transferusing a Northern/Southern Transfer buffer system (TEL-TEST “B” Inc.,Friendswood, Tex.). RNA transfer was confirmed by UV visualization.Membranes were fixed by UV cross-linking using a STRATALINKER™ UVCrosslinker 2400 (Stratagene, Inc, La Jolla, Calif.) and then probedusing QUICKHYB™ hybridization solution (Stratagene, La Jolla, Calif.)using manufacturer's recommendations for stringent conditions.

[0270] To detect human hypothetical tumor endothelial marker, a humanhypothetical tumor endothelial marker specific probe was prepared by PCRusing the forward primer CTGGGCCACATCAGAATAAGG (SEQ ID NO: 5) and thereverse primer CGCAATCAGGACAGTCAGCAT (SEQ ID NO: 6). To normalize forvariations in loading and transfer efficiency membranes were strippedand probed for human glyceraldehyde-3-phosphate dehydrogenase (GAPDH)RNA (Clontech, Palo Alto, Calif.).

[0271] Hybridized membranes were visualized and quantitated using aPHOSPHORIMAGER™ and IMAGEQUANT™ Software V3.3 (Molecular Dynamics,Sunnyvale, Calif.). Data was normalized to GAPDH levels in untreatedcontrols.

Example 15

[0272] Antisense Inhibition of Human Hypothetical Tumor EndothelialMarker Expression by Chimeric Phosphorothioate Oligonucleotides Having2′-MOE Wings and a Deoxy Gap

[0273] In accordance with the present invention, a series ofoligonucleotides were designed to target different regions of the humanhypothetical tumor endothelial marker RNA, using published sequences(GenBank accession number AB062750.1, incorporated herein as SEQ ID NO:4, and the complement of residues 10000-105000 of GenBank accessionnumber AC073341.9, representing a genomic sequence of hypothetical tumorendothelial marker, incorporated herein as SEQ ID NO: 11). Theoligonucleotides are shown in Table 1. “Target site” indicates the first(5′-most)nucleotide number on the particular target sequence to whichthe oligonucleotide binds. All compounds in Table 1 are chimericoligonucleotides (“gapmers”) 20 nucleotides in length, composed of acentral “gap” region consisting of ten 2′-deoxynucleotides, which isflanked on both sides (5′ and 3′directions) by five-nucleotide “wings”.The wings are composed of 2′-methoxyethyl (2′-MOE)nucleotides. Theinternucleoside (backbone) linkages are phosphorothioate (P═S)throughout the oligonucleotide. All cytidine residues are5-methylcytidines. The compounds were analyzed for their effect on humanhypothetical tumor endothelial marker mRNA levels by quantitativereal-time PCR as described in other examples herein. Data are averagesfrom two experiments in which A549 cells were treated with theoligonucleotides of the present invention. The positive control for eachdatapoint is identified in the table by sequence ID number. If present,“N.D.” indicates “no data”. TABLE 1 Inhibition of human hypotheticaltumor endothelial marker mRNA levels by chimeric phosphorothioateoligonucleotides having 2′-MOE wings and a deoxy gap TARGET CONTROL SEQID TARGET SEQ ID SEQ ID ISIS # REGION NO SITE SEQUENCE % INHIB NO NO208361 5′UTR 4 63 tcagcttcctgagtctcccg 90 12 1 208362 5′UTR 4 89agcatcgttgtcgtactgcc 81 13 1 208363 5′UTR 4 122 ccatgcacatttggagaagg 8914 1 208364 5′UTR 4 129 cctttccccatgcacatttg 58 15 1 208365 Start 4 198gggtcatcgtctctttgacg 56 16 1 Codon 208366 Coding 4 242taaaattctgccatcgataa 83 17 1 208367 Coding 4 292 tctggagacacaggaaatgc 4718 1 208368 Coding 4 313 gctgttttcacatacggtgt 34 19 1 208369 Coding 4403 gggcagcttcgtggttcacg 80 20 1 208370 Coding 4 465gcatcgtggatttgggtccg 80 21 1 208371 Coding 4 505 gcctgctgaaaggagggcgt 4422 1 208372 Coding 4 531 tggaaatggtgcaggaagaa 79 23 1 208373 Coding 4680 gtccttcctggtaccggaga 80 24 1 208374 Coding 4 747gggacagctcatggctgtgg 83 25 1 208375 Coding 4 859 agcaaggcccccaggtcctc 7926 1 208376 Coding 4 938 ggacaggaagccattgtcgg 51 27 1 208377 Coding 4979 tggtggctgctgtgtccagg 94 28 1 208378 Coding 4 1114ctgtgcgggctggagaagcc 68 29 1 208379 Coding 4 1182 Aggccgcttctgaagccttg80 30 1 208380 Coding 4 1291 atggcgatggcttgttctct 46 31 1 208381 Coding4 1437 cgagttcattggccaaatct 83 32 1 208382 Coding 4 1504tatggttcattcgagcaccc 69 33 1 208383 Coding 4 1562 cggcaaggccaagggcgtga88 34 1 208384 Coding 4 1597 tccaatggatctctctctgg 81 35 1 208385 Coding4 1697 ctccacagagttcaagtacc 76 36 1 208386 Coding 4 1746tgatgctcagggccttctgg 89 37 1 208387 Coding 4 1857 ggtaatgcctccggaagaag60 38 1 208388 Coding 4 1917 catctttgatccacttcctg 36 39 1 208389 Coding4 2021 actggcaggctgctcagggt 93 40 1 208390 Stop 4 2083gggagttctcagaccttctt 69 41 1 Codon 208391 3′UTR 4 2132acggctgtctccagggcttc 94 42 1 208392 3′UTR 4 2281 tgtcagataagtcactttca 8743 1 208393 3′UTR 4 2318 gcttcttctacccaaaaagc 73 44 1 208394 3′UTR 42477 atggcagtcactcggcacct 92 45 1 208395 3′UTR 4 2518gggtgcagctggacagaagc 83 46 1 208396 3′UTR 4 2588 tacgcccccttctcagtttc 7847 1 208397 3′UTR 4 2619 gggcctgacttgcacaaacc 90 48 1 208398 3′UTR 42704 ctaacgtggccaattcttcc 88 49 1 208399 3′UTR 4 2745gaggcacagagcactcacca 89 50 1 208400 3′UTR 4 2940 aggctgcagcatttgacact 8251 1 208401 3′UTR 4 2990 cctgcggtgoatcctacata 96 52 1 208402 3′UTR 43025 cacagtccactgcacactct 56 53 1 208403 3′UTR 4 3096caatgagtaaaacatcaggg 80 54 1 208404 3′UTR 4 3109 gggaaaaacattccaatgag 7255 1 208405 3′UTR 4 3211 gaagatgatcgaaaagccca 77 56 1 208406 3′UTR 43309 ccacacatggtccccagcag 90 57 1 208407 3′UTR 4 3385cactcaaaaacagcttgtca 81 58 1 208408 3′UTR 4 3507 gtcacaaattcccccaaacc 6259 1 208409 3′UTR 4 3613 caggacagtcagcatgcctg 79 60 1 208410 3′UTR 43738 cagagttcacaaagcacatt 56 61 1 208411 3′UTR 4 3803cactgtgatttggctgcttc 92 62 1 208412 3′UTR 4 3875 aagtggaatggtccacctgc 8363 1 208413 3′UTR 4 4011 atgaggatctgctgaaaatg 71 64 1 208414 3′UTR 44054 ccgagatgctagaggcctct 89 65 1 208415 3′UTR 4 4219gctctgacttctaaatgagc 85 66 1 208416 3′UTR 4 4256 tggcaggctgcgtttctcaa 9367 1 208417 3′UTR 4 4262 attccctggcaggctgcgtt 96 68 1 208418 3′UTR 44347 Tcaaaaagcacgtttgcagg 89 69 1 208419 3′UTR 4 4427ggagctatctgcttggagca 86 70 1 208420 3′UTR 4 4621 acgttccttttcctgtggcc 9371 1 208421 3′UTR 4 4685 tgaactctccgcatttactc 50 72 1 208422 3′UTR 44715 ctggctataacatgcgttgg 84 73 1 208423 3′UTR 4 4756aatacaacaatacgttcttt 43 74 1 208424 3′UTR 4 4780 atggctatagcttaacactg 8575 1 208425 3′UTR 4 4795 acagtgacttttaacatggc 89 76 1 208426 3′UTR 44808 tgagaataaatgcacagtga 83 77 1 208427 3′UTR 4 4884atatattctttacagtatca 88 78 1 208428 3′UTR 4 4911 tttatagaacattcatttac 379 1 208429 3′UTR 4 4965 atactctgaatagagatgat 74 80 1 208430 3′UTR 44984 ccaagttcataattttatta 25 81 1 208431 Intron 11 1442ccttcagccccactgaagta 81 82 1 208432 Exon: 11 24455 tcattcttaccatacggtgt38 83 1 Intron Junction 208433 Exon: 11 24624 gatttcttaccacgtcctcc 47 841 Intron Junction 208434 Intron 11 40376 tgctgaaaaactcctataca 69 85 1208435 Intron 11 60232 ctttcactgcctgatgccca 86 86 1 208436 Intron 1161676 actaatgatgttgaacatct 75 87 1 208437 Intron: 11 89188Tttgatccaccttgcaaatt 53 88 1 Exon Junction 208438 Intron: 11 91158tccaaagactctgccaaagg 66 89 1 Exon Junction

[0274] As shown in Table 1, SEQ ID NOs 12, 13, 14, 17, 20, 21, 23, 24,25, 26, 28, 29, 30, 32, 33, 34, 35, 36, 37, 40, 41, 42, 43, 44, 45, 46,47, 48, 49, 50, 51, 52, 54, 55, 56, 57, 58, 59, 60, 62, 63, 64, 65, 66,67, 68, 69, 70, 71, 73, 75, 76, 77, 78, 80, 82, 85, 86, 87 and 89demonstrated at least 62% inhibition of human hypothetical tumorendothelial marker expression in this assay and are therefore preferred.The target sites to which these preferred sequences are complementaryare herein referred to as “preferred target regions” and are thereforepreferred sites for targeting by compounds of the present invention.These preferred target regions are shown in Table 2. The sequencesrepresent the reverse complement of the preferred antisense compoundsshown in Table 1. “Target site” indicates the first (5′-most) nucleotidenumber of the corresponding target nucleic acid. Also shown in Table 2is the species in which each of the preferred target regions was found.TABLE 2 Sequence and position of preferred target regions identified inhypothetical tumor endothelial marker. TARGET TARGET REV COMP SEQ IDSITEID SEQ ID NO SITE SEQUENCE OF SEQ ID ACTIVE IN NO 125975 4 63cgggagactcaggaagctga 12 H. sapiens 90 125976 4 89 ggcagtacgacaacgatgct13 H. sapiens 91 125977 4 122 ccttctceaaatgtgcatgg 14 H. sapiens 92125980 4 242 ttatcgatggcagaatttta 17 H. sapiens 93 125983 4 403cgtgaaccacgaagctgccc 20 H. sapiens 94 125984 4 465 cggacccaaatccacgatgc21 H. sapiens 95 125986 4 531 ttcttcctgcaccatttcca 23 H. sapiens 96125987 4 680 tctccggtaccaggaaggac 24 H. sapiens 97 125988 4 747ccacagccatgagctgtccc 25 H. sapiens 98 125989 4 859 gaggacctgggggccttgct26 H. sapiens 99 125991 4 979 cctggacacagcagccacca 28 H. sapiens 100125992 4 1114 ggcttctccagcccgcacag 29 H. sapiens 101 125993 4 1182caaggcttcagaagcggcct 30 H. sapiens 102 125995 4 1437agatttggccaatgaactcg 32 H. sapiens 103 125996 4 1504gggtgctcgaatgaaccata 33 H. sapiens 104 125997 4 1562tcacgcccttggccttgccg 34 H. sapiens 105 125998 4 1597ccagagagagatccattgga 35 H. sapiens 106 125999 4 1697ggtacttgaactctgtggag 36 H. sapiens 107 126000 4 1746ccagaaggccctgagcatca 37 H. sapiens 108 126003 4 2021accctgagcagcctgccagt 40 H. sapiens 109 126004 4 2083aagaaggtctgagaactccc 41 H. sapiens 110 126005 4 2132gaagccctggagacagccgt 42 H. sapiens 111 126006 4 2281tgaaagtgacttatctgaca 43 H. sapiens 112 126007 4 2318gctttttgggtagaagaagc 44 H. sapiens 113 126008 4 2477aggtgccgagtgactgccat 45 H. sapiens 114 126009 4 2518gcttctgtccagctgcaccc 46 H. sapiens 115 126010 4 2588gaaactgagaagggggcgta 47 H. sapiens 116 126011 4 2619ggtttgtgcaagtcaggccc 48 H. sapiens 117 126012 4 2704ggaagaattggccacgttag 49 H. sapiens 118 126013 4 2745tggtgagtgctctgtgcctc 50 H. sapiens 119 126014 4 2940agtgtcaaatgctgcagcct 51 H. sapiens 120 126015 4 2990tatgtaggatgcaccgcagg 52 H. sapiens 121 126017 4 3096ccctgatgttttactcattg 54 H. sapiens 122 126018 4 3109ctcattggaatgtttttccc 55 H. sapiens 123 126019 4 3211tgggcttttcgatcatcttc 56 H. sapiens 124 126020 4 3309ctgctggggaccatgtgtgg 57 H. sapiens 125 126021 4 3385tgacaagctgtttttgagtg 58 H. sapiens 126 126022 4 3507ggtttgggggaatttgtgac 59 H. sapiens 127 126023 4 3613caggcatgctgactgtcctg 60 H. sapiens 128 126025 4 3803gaagcagccaaatcacagtg 62 H. sapiens 129 126026 4 3875gcaggtggaccattccactt 63 H. sapiens 130 126027 4 4011cattttcagcagatcctcat 64 H. sapiens 131 126028 4 4054agaggcctctagcatctcgg 65 H. sapiens 132 126029 4 4219gctcatttagaagtcagagc 66 H. sapiens 133 126030 4 4256ttgagaaacgcagcctgcca 67 H. sapiens 134 126031 4 4262aacgcagcctgccagggaat 68 H. sapiens 135 126032 4 4347cctgcaaacgtgctttttga 69 H. sapiens 136 126033 4 4427tgctccaagcagatagctcc 70 H. sapiens 137 126034 4 4621ggccacaggaaaaggaacgt 71 H. sapiens 138 126036 4 4715ccaacgcatgttatagccag 73 H. sapiens 139 126038 4 780 cagtgttaagctatagccat75 H. sapiens 140 126039 4 4795 gccatgttaaaagtcactgt 76 H. sapiens 141126040 4 4808 tcactgtgcatttattctca 77 H. sapiens 142 126041 4 4884tgatactgtaaagaatatat 78 H. sapiens 143 126043 4 4965atcatctctattcagagtat 80 H. sapiens 144 126045 11 1442tacttcagtggggctgaagg 82 H. sapiens 145 126048 11 40376tgtataggagtttttcagca 85 H. sapiens 146 126049 11 60232tgggcatcaggcagtgaaag 86 H. sapiens 147 126050 11 61676agatgttcaacatcattagt 87 H. sapiens 148 126052 11 91158cctttggcagagtctttgga 89 H. sapiens 149

[0275] As these “preferred target regions” have been found byexperimentation to be open to, and accessible for, hybridization withthe antisense compounds of the present invention, one of skill in theart will recognize or be able to ascertain, using no more than routineexperimentation, further embodiments of the invention that encompassother compounds that specifically hybridize to these sites andconsequently inhibit the expression of hypothetical tumor endothelialmarker.

[0276] In one embodiment, the “preferred target region” may be employedin screening candidate antisense compounds. “Candidate antisensecompounds” are those that inhibit the expression of a nucleic acidmolecule encoding hypothetical tumor endothelial marker and whichcomprise at least an 8-nucleobase portion which is complementary to apreferred target region. The method comprises the steps of contacting apreferred target region of a nucleic acid molecule encoding hypotheticaltumor endothelial marker with one or more candidate antisense compounds,and selecting for one or more candidate antisense compounds whichinhibit the expression of a nucleic acid molecule encoding hypotheticaltumor endothelial marker. Once it is shown that the candidate antisensecompound or compounds are capable of inhibiting the expression of anucleic acid molecule encoding hypothetical tumor endothelial marker,the candidate antisense compound may be employed as an antisensecompound in accordance with the present invention.

[0277] According to the present invention, antisense compounds includeribozymes, external guide sequence (EGS) oligonucleotides (oligozymes),and other short catalytic RNAs or catalytic oligonucleotides whichhybridize to the target nucleic acid and modulate its expression.

Example 16

[0278] Western Blot Analysis of Hypothetical Tumor Endothelial MarkerProtein Levels

[0279] Western blot analysis (immunoblot analysis) is carried out usingstandard methods. Cells are harvested 16-20 h after oligonucleotidetreatment, washed once with PBS, suspended in Laemmli buffer (100ul/well), boiled for 5 minutes and loaded on a 16% SDS-PAGE gel. Gelsare run for 1.5 hours at 150 V, and transferred to membrane for westernblotting. Appropriate primary antibody directed to hypothetical tumorendothelial marker is used, with a radiolabeled or fluorescently labeledsecondary antibody directed against the primary antibody species. Bandsare visualized using a PHOSPHORIMAGER™ (Molecular Dynamics, SunnyvaleCalif.).

1 149 1 20 DNA Artificial Sequence Antisense Oligonucleotide 1tccgtcatcg ctcctcaggg 20 2 20 DNA Artificial Sequence AntisenseOligonucleotide 2 gtgcgcgcga gcccgaaatc 20 3 20 DNA Artificial SequenceAntisense Oligonucleotide 3 atgcattctg cccccaagga 20 4 5076 DNA H.sapiens CDS (211)...(2094) 4 acaggagagg gccccagcag ggccaccggg cggcaaggctcctctgctga acagcccctg 60 ggcgggagac tcaggaagct gagcctgggg cagtacgacaacgatgctgg ggggcagctg 120 cccttctcca aatgtgcatg gggaaaggct ggtgtggactatgccccaaa cctgccgcca 180 ttcccctcac cagcggacgt caaagagacg atg acc cctggc tat ccc cag gac 234 Met Thr Pro Gly Tyr Pro Gln Asp 1 5 ctc gat attatc gat ggc aga att tta agt agc aag gag tcc atg tgt 282 Leu Asp Ile IleAsp Gly Arg Ile Leu Ser Ser Lys Glu Ser Met Cys 10 15 20 tca act cca gcattt cct gtg tct cca gag aca ccg tat gtg aaa aca 330 Ser Thr Pro Ala PhePro Val Ser Pro Glu Thr Pro Tyr Val Lys Thr 25 30 35 40 gcg ctg cgc catcct ccg ttc agc cca cct gag ccc ccg ctg agc agc 378 Ala Leu Arg His ProPro Phe Ser Pro Pro Glu Pro Pro Leu Ser Ser 45 50 55 cca gcc agt cag cacaaa gga gga cgt gaa cca cga agc tgc cct gag 426 Pro Ala Ser Gln His LysGly Gly Arg Glu Pro Arg Ser Cys Pro Glu 60 65 70 acg ctc act cac gct gtgggg atg tca gag agc ccc atc gga ccc aaa 474 Thr Leu Thr His Ala Val GlyMet Ser Glu Ser Pro Ile Gly Pro Lys 75 80 85 tcc acg atg ctc cgg gct gatgcg tcc tcg acg ccc tcc ttt cag cag 522 Ser Thr Met Leu Arg Ala Asp AlaSer Ser Thr Pro Ser Phe Gln Gln 90 95 100 gct ttt gct tct tcc tgc accatt tcc agc aac ggc cct ggg cag agg 570 Ala Phe Ala Ser Ser Cys Thr IleSer Ser Asn Gly Pro Gly Gln Arg 105 110 115 120 aga gag agc tcc tct tctgca gaa cgc cag tgg gtg gag agc agc ccc 618 Arg Glu Ser Ser Ser Ser AlaGlu Arg Gln Trp Val Glu Ser Ser Pro 125 130 135 aag ccc atg gtt tcc ctgctg ggg agc ggc cgg ccc acc gga agt ccc 666 Lys Pro Met Val Ser Leu LeuGly Ser Gly Arg Pro Thr Gly Ser Pro 140 145 150 ctc agc gct gag ttc tccggt acc agg aag gac tcc cca gtg ctg tcc 714 Leu Ser Ala Glu Phe Ser GlyThr Arg Lys Asp Ser Pro Val Leu Ser 155 160 165 tgc ttc ccg ccg tca gagctc cag gct cct ttc cac agc cat gag ctg 762 Cys Phe Pro Pro Ser Glu LeuGln Ala Pro Phe His Ser His Glu Leu 170 175 180 tcc cta gca gag cca ccggac tcc ctg gcg cct ccc agc agc cag gcc 810 Ser Leu Ala Glu Pro Pro AspSer Leu Ala Pro Pro Ser Ser Gln Ala 185 190 195 200 ttc ctg ggc ttc ggcacc gcc cca gtg gga agt ggc ctt ccg ccc gag 858 Phe Leu Gly Phe Gly ThrAla Pro Val Gly Ser Gly Leu Pro Pro Glu 205 210 215 gag gac ctg ggg gccttg ctg gcc aat tct cat gga gcg tca ccg acc 906 Glu Asp Leu Gly Ala LeuLeu Ala Asn Ser His Gly Ala Ser Pro Thr 220 225 230 ccc agc atc ccg ctgaca gcg aca ggg gct gcc gac aat ggc ttc ctg 954 Pro Ser Ile Pro Leu ThrAla Thr Gly Ala Ala Asp Asn Gly Phe Leu 235 240 245 tcc cac aac ttt ctcacg gtg gcg cct gga cac agc agc cac cac agt 1002 Ser His Asn Phe Leu ThrVal Ala Pro Gly His Ser Ser His His Ser 250 255 260 cca ggc ctg cag ggccag ggt gtg acc ctg ccc ggg cag cca ccc ctc 1050 Pro Gly Leu Gln Gly GlnGly Val Thr Leu Pro Gly Gln Pro Pro Leu 265 270 275 280 cct gag aag aagcgg gcc tcg gag ggg gat cgt tct ttg ggc tca gtc 1098 Pro Glu Lys Lys ArgAla Ser Glu Gly Asp Arg Ser Leu Gly Ser Val 285 290 295 tct ccc tcc tccagt ggc ttc tcc agc ccg cac agc ggg agc acc atc 1146 Ser Pro Ser Ser SerGly Phe Ser Ser Pro His Ser Gly Ser Thr Ile 300 305 310 agt atc ccc ttccca aat gtc ctt ccc gac ttt tcc aag gct tca gaa 1194 Ser Ile Pro Phe ProAsn Val Leu Pro Asp Phe Ser Lys Ala Ser Glu 315 320 325 gcg gcc tca cctctg cca gat agt cca ggt gat aaa ctt gtg atc gtg 1242 Ala Ala Ser Pro LeuPro Asp Ser Pro Gly Asp Lys Leu Val Ile Val 330 335 340 aaa ttt gtt caagac act tcc aag ttc tgg tac aag gcg gat att tca 1290 Lys Phe Val Gln AspThr Ser Lys Phe Trp Tyr Lys Ala Asp Ile Ser 345 350 355 360 aga gaa caagcc atc gcc atg ttg aag gac aag gag ccg ggc tca ttc 1338 Arg Glu Gln AlaIle Ala Met Leu Lys Asp Lys Glu Pro Gly Ser Phe 365 370 375 att gtt cgagac agc cat tcc ttc cga ggg gcc tat ggc ctg gcc atg 1386 Ile Val Arg AspSer His Ser Phe Arg Gly Ala Tyr Gly Leu Ala Met 380 385 390 aag gtg gccacg ccc cca cct tca gtc ctg cag ctg aac aag aaa gct 1434 Lys Val Ala ThrPro Pro Pro Ser Val Leu Gln Leu Asn Lys Lys Ala 395 400 405 gga gat ttggcc aat gaa ctc gtc cgg cac ttt ttg atc gag tgt acc 1482 Gly Asp Leu AlaAsn Glu Leu Val Arg His Phe Leu Ile Glu Cys Thr 410 415 420 ccg aag ggagtg cgg ttg aaa ggg tgc tcg aat gaa cca tat ttc ggg 1530 Pro Lys Gly ValArg Leu Lys Gly Cys Ser Asn Glu Pro Tyr Phe Gly 425 430 435 440 agc ctgacg gcc ttg gtg tgc cag cat tcc atc acg ccc ttg gcc ttg 1578 Ser Leu ThrAla Leu Val Cys Gln His Ser Ile Thr Pro Leu Ala Leu 445 450 455 ccg tgcaag ctg ctt atc cca gag aga gat cca ttg gag gaa ata gca 1626 Pro Cys LysLeu Leu Ile Pro Glu Arg Asp Pro Leu Glu Glu Ile Ala 460 465 470 gaa agttct ccc cag acg gca gcc aat tca gca gct gag ctg ttg aag 1674 Glu Ser SerPro Gln Thr Ala Ala Asn Ser Ala Ala Glu Leu Leu Lys 475 480 485 cag ggggca gcc tgc aac gtg tgg tac ttg aac tct gtg gag atg gag 1722 Gln Gly AlaAla Cys Asn Val Trp Tyr Leu Asn Ser Val Glu Met Glu 490 495 500 tcc ctcacc ggc cac cag gcg atc cag aag gcc ctg agc atc acc ctg 1770 Ser Leu ThrGly His Gln Ala Ile Gln Lys Ala Leu Ser Ile Thr Leu 505 510 515 520 gtccag gag cct cca cct gtg tcc aca gtt gtg cac ttc aag gtg tca 1818 Val GlnGlu Pro Pro Pro Val Ser Thr Val Val His Phe Lys Val Ser 525 530 535 gcccag ggc atc acc ctg aca gac aat cag agg aag ctc ttc ttc cgg 1866 Ala GlnGly Ile Thr Leu Thr Asp Asn Gln Arg Lys Leu Phe Phe Arg 540 545 550 aggcat tac ccc gtg aac agt gtg att ttc tgt gcc ttg gac cca caa 1914 Arg HisTyr Pro Val Asn Ser Val Ile Phe Cys Ala Leu Asp Pro Gln 555 560 565 gacagg aag tgg atc aaa gat ggc cct tcc tca aaa gtc ttt gga ttt 1962 Asp ArgLys Trp Ile Lys Asp Gly Pro Ser Ser Lys Val Phe Gly Phe 570 575 580 gtggcc cgg aag cag ggc agt gcc acg gat aat gtg tgc cac ctg ttt 2010 Val AlaArg Lys Gln Gly Ser Ala Thr Asp Asn Val Cys His Leu Phe 585 590 595 600gca gag cat gac cct gag cag cct gcc agt gcc att gtc aac ttc gta 2058 AlaGlu His Asp Pro Glu Gln Pro Ala Ser Ala Ile Val Asn Phe Val 605 610 615tca aag gtc atg att ggt tcc cca aag aag gtc tga gaactcccct 2104 Ser LysVal Met Ile Gly Ser Pro Lys Lys Val 620 625 ccctccctgg acccaccgatgcctctcgaa gccctggaga cagccgttgg gtgagggtgg 2164 ggcccccact ttttaccaaactagtaaacc tgacattcca ggcccatgag gggaaagagg 2224 atcttccagc tctgcaaaaacaagaacaaa caacatcacc gtgaattggc ctttcctgaa 2284 agtgacttat ctgacacatctctgtagcca catgcttttt gggtagaaga agctgggcat 2344 gggtgcaccc caccccctagggtccccatg ggaaagggac atgcaaggaa acagcacaga 2404 acacgaggtg gtccccatgtccctggcaca ctagcattct gggggatgag gaatccccag 2464 cccttgaggc agaggtgccgagtgactgcc atgcttcgcc cgtccgcatg ggcgcttctg 2524 tccagctgca cccgaggccgggggtttccc tcacctcggt cttcccaaga tggagatgct 2584 aacgaaactg agaagggggcgtatgtttga cgaaggtttg tgcaagtcag gcccttctgg 2644 aacacagcag ggcctacaacgaggggcctt tgcgatgggc tgtgaggatg ggggtggtgg 2704 gaagaattgg ccacgttagagaccccatgc caccccacca tggtgagtgc tctgtgcctc 2764 ctgctcacct gtggtgagctgggcgagctg ggcgagctgg gcgagctggg ctggggagag 2824 cctgtgagga ccgagaggagaaatgagaag aaggaacaaa aatattattt ctatgtaatt 2884 tatattttac ttatgccaaattatttatga taatttgcca ttgctatact gtaccagtgt 2944 caaatgctgc agcctgccaagctgtgattt tgtgaggctt gtccctatgt aggatgcacc 3004 gcaggcccct ggccactgaaagagtgtgca gtggactgtg ggtctcccat atgcggtgcc 3064 gcccaaaggt ggctttgcctcaagcaacct accctgatgt tttactcatt ggaatgtttt 3124 tccccgattg tggatgacttcttttctgat ggagagagtc caggagggat ggaaaactcc 3184 tggatttaag ctcagcatcccccacatggg cttttcgatc atcttcaggc ctgaagctgc 3244 acgacctgaa gttcgcctgcatttatcagc cctctttgtg ctgctccttg ccaccttggg 3304 gttcctgctg gggaccatgtgtggttgtgg catgtgtgag cagaagggag gatgaggaaa 3364 aagagaagaa accccggtactgacaagctg tttttgagtg ccactgtttg ccatcatcta 3424 agccactgaa tcaagtgtatttcaggctta tttcaacatt ccaatgccct ggttttcctg 3484 cttgaatctg ttcgtggtcaaaggtttggg ggaatttgtg accctggaac atccccagag 3544 tgaaagatgg agctgggccacatcagaata aggccttggc cccatcctct cacagcctag 3604 gtgctctgca ggcatgctgactgtcctgat tgcgatccag cccgaaattc cctcctctgc 3664 tttcaaaagt caaatcccccattcttaggc cacactggtg tcacaagctc ctgtcaggga 3724 gctggggttt gggaatgtgctttgtgaact ctgctttaaa gtgaggggcc gaggaaaact 3784 tagaaacagg cagagttggaagcagccaaa tcacagtggg tgttgtgtgt gtgtgcgtgt 3844 gtgcatgcgt gcgtgtatgcgtgtgtgaaa gcaggtggac cattccactt tttagctcct 3904 attgatgcac caaaccaagtgcctcatttc tgtgccaaat gtttgccttg gtcgttgtgg 3964 acctccttct ctaacttgcggtggcatgac tgtcaggagg tgctggcatt ttcagcagat 4024 cctcatgtgt tgaccctgatgtctttagca gaggcctcta gcatctcggt ttttcatcca 4084 ctgcaggaat gtggccacagggagcagagg tttgtacttt ccccaagagg tcctcatcct 4144 gagacggtct ctacccatgtttaacccaaa gagtgcaggc caggttcctt atccttctga 4204 tgaaggatga gagagctcatttagaagtca gagcaaacta gggtctcagt attgagaaac 4264 gcagcctgcc agggaatcacagagacatcg gggtgcccgc gatggccctc atgaagccat 4324 gcctcgacgg cattcaggaagccctgcaaa cgtgcttttt gaactcattg gccaggtgtg 4384 atttttacac aaggtaaacgtggtcaaggg catcggggaa tttgctccaa gcagatagct 4444 ccctctgagg aaccaaaggaagcaagtttc catgatttct gaagagctgg tataggaagt 4504 ttctttcttc cttttgtgttacatgtgcat taaacagaac aagctgtgtg tcatcacaga 4564 ttgtactgtg ggctcagaaaccgtgagaga gcccccaccg tggacaccgg ctctagggcc 4624 acaggaaaag gaacgtttccaggcattttg tctccatgga caggcacgta ctgccctggg 4684 gagtaaatgc ggagagttcacgaactgtgc ccaacgcatg ttatagccag ggtcctacta 4744 actactcagt aaaagaacgtattgttgtat tcctccagtg ttaagctata gccatgttaa 4804 aagtcactgt gcatttattctcagcatcaa ataccttgta acgtcttctc tgccttgtta 4864 gtgcatattt ttacttttctgatactgtaa agaatatatc cagtatgtaa atgaatgttc 4924 tataaatctt ttgtatagtcattttctctg ctccttaaat atcatctcta ttcagagtat 4984 aataaaatta tgaacttggtaaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 5044 aaaaaaaaaa aaaaaaaaaaaaaaaaaaaa aa 5076 5 21 DNA Artificial Sequence PCR Primer 5 ctgggccacatcagaataag g 21 6 21 DNA Artificial Sequence PCR Primer 6 cgcaatcaggacagtcagca t 21 7 26 DNA Artificial Sequence PCR Probe 7 cttggccccatcctctcaca gcctag 26 8 19 DNA Artificial Sequence PCR Primer 8gaaggtgaag gtcggagtc 19 9 20 DNA Artificial Sequence PCR Primer 9gaagatggtg atgggatttc 20 10 20 DNA Artificial Sequence PCR Probe 10caagcttccc gttctcagcc 20 11 95001 DNA Homo sapiens 11 ccaggagaaggcagagttgg accagctgct cagtggcttt ggcctggaag atcctggaag 60 ctccctcaaggaaatgactg atgctcgaag caagtacagt gggacccgcc acgtggtgcc 120 agcccaggttcacgtgaatg gagacgctgc tctgaaggat cgggagacag acattctgga 180 tgacgagatgccccaccacg acctgcacag tgtggacagc cttgggaccc tgtcctcctc 240 ggaagggcctcagtcggcca cctgggtccc ttcacctgcc acaagagcag ccagaactca 300 ctcctatctgacggttttgg cagcaacgtt ggtgaagatc cgcagggcac cctcgttccg 360 gacctgggccttggcatgga cggcccctat gagcgggagc ggacttttgg gagtcgagag 420 cccaagcagccccagcccct gctgagaaag ccctcagtgt ccgcccagat gcaggcctat 480 gggcagagcagctactccac acagacctgg gtgcgccagc agcagatggt tgtagctcac 540 cagtatagcttcgccccaga tggggaggcc cggctggtga gccgctgccc tgcagacaat 600 cctggcctcgtccaggccca gcccagagtg ccactcaccc ccacccgagg gaccagcagt 660 agggtggctgtccagagggg tgtaggcagt gggccacatc cccctgacac acagcagccc 720 tctcccagcaaagcgttcaa acccaggttt ccaggagacc aggttgtgaa tggagccggc 780 ccagagctgagcacaggccc ctccccaggc tcgcccaccc tggacatcga ccagtccatc 840 gagcagctcaacaggctgat cctggagctg gatcccacct tcgagcccat ccctacccac 900 atgaacgccctcggtagcca ggccaatggc tctgtgtctc cagacagcgt gggaggtggg 960 ctccgggcaagcagcaggct gcctgacaca ggagagggcc ccagcagggc caccgggcgg 1020 caaggtgagctgggtctcca tgggaggtgc tccacgggag gtgctccacg ggaggctaat 1080 gtgtgatgagaaatcagcct agtaggttag cttttggcga aatccacaag ggagggacat 1140 ttcccttgcagtttcaactc ttgtgaattt tggcctagga tccaaatgct gtcatgctgg 1200 tgtggtcccttggtaggaga tttggaggat gggaagaacc aggcagaaga cttgctgtta 1260 acgtcagtgcaggggcaggg acagcatgct ctctgctgga aagggccggt catgcctcca 1320 ttgcagaatgggcttaacag tgaaaccatt cttccttcgt gaatgttggt ggatttacaa 1380 agaaagcatacttacttctg tccctgcggt ataggtcagg gctttgtgga agaatgccag 1440 gtacttcagtggggctgaag gttggggatg aagaagacta ctgagccctc agacagcttt 1500 ggcacaaggaattgattaag cagtcgtggt ataaaatgtt ctgattacaa atacacctgt 1560 cagagttggccgtggtggtg tggggagact ggttatggaa ggcagtggct gggcagaggc 1620 ctggggtccacaaggggttc tactgtggag agcagctcag tgatgggagg ggcttggggg 1680 aacaggcccaggggtgaggg gactgagagc tgtggtggga cccaaagcca tgggacccgg 1740 tgcggggcctctggggatgc cccaaaacca ggccggggtc atgctaactg cagggaggcc 1800 aggactgcacatgtcctttt cccgtgaggc tggtggtttg gaaaccagca cagtgccttt 1860 ctgtgcagccttgaggcctg ggaggaacca gggctgagag tggtggacag gcagtgacat 1920 ctgcctgggttacagcagca ctatcttcaa gctgcatcga tgtaatgggg caagttgcat 1980 gttgggaccaggttactata cagtgttctt tctgttggga aagcatgaag gctgagggga 2040 ccagagagctggaggtggtc aggtcggcca gggaaacact cagcaggcag agaggggcat 2100 tgggacggagcagggttgtc tcaggtaggg gactgtgtgg gtgagatcat gtggtctgga 2160 agctcctgcacagaggtgca cgccaggaga ggacggccag gctgtagcgg ctgtgcctgc 2220 gggcactgggaggctacaga aggtggagag acagagctgt gttctgggag ccctggcagt 2280 gccatgcaaggaccaggacc caaaggataa ccttgggcca gaaagtgtgg catccgtgtg 2340 gtgccagggcctcccacatt ggcacgttgg ggtgttttgc catctggggg ctgctggctc 2400 cttagagggacttgcctcta aaagtgaaga ccagagaggt aaaggacaag ggacatggct 2460 gtgtgacgccgtgcatgtga ttcccaccat gtgctggtaa cacggatgag agatgctacc 2520 atttatgctggcgtggttct tggtttaggt gtgctggccc atgtcatcgt cctaagcacc 2580 ccggctgcaggcaccagtct ctgccttaca gtcgtgttga tggaggtgtc agtagattat 2640 gtggcctgcccaggttcaca tgggacctgc atagtagagc aaaagtcagt gccctgaggt 2700 gtggactcagtgtctccatc ctctttggag tcacacaggt gaggactgca gtcccgactc 2760 aggtctggggtggcctgggc gaaggagcac acaccctgca gatgttggtc ctggcttcag 2820 gtctgagtgtgctgtgggtt gcctgagtga tgcaagtggg taaagggcag cgtctctgtg 2880 aatttctcttcactccatct gtgagaagga aaccctatcc cagtgcctgg cccggggaag 2940 gtgtgaagaaacgctcgttc acatccacct ttcatgtact aggtgtgtga ctgagcaaac 3000 agctgtcattccagcttgcc tgagtaactc cctctttgta tgtcatgtgc tgggagttgg 3060 aaggagcctggagagtcccc catgagtaga ttattggact ttacaataca gccgtatagc 3120 aaccacaacaccacagcagg gtagcagtcc ctgaacaata ggtccaggtc gatgcattaa 3180 acaaatatatttgggaaatt ctgagtttac taaaaaggct ctaatgggaa aaaactatct 3240 gcagtatcatacctgactta cttcaccaaa ccttcttaaa agaagttact tggaaacatt 3300 aaaatcagaagtcttttctc ctggataaca atctcatgta ggtttggagg aggctggagg 3360 catctgagtgtatgtggaga agaggactcc acatacttag gttaacatat agtagtctct 3420 tggcttaggttaacatatag tagtctcttg aggttaatat gtcttccaat tgattgaatt 3480 caatccttagtgattccctg ttcttgctgc aaagaaacag ttataaggcc tggaattttc 3540 gaaagccacatcatattatc caccctcagt ttggctgttt ttaaattact tggtaaactc 3600 attactttatttttttattt atttttttga gacagagtct cactctgtcg cccaagctgg 3660 agtgcaatggcgtgatctca gcttactgca agctccacct cccgggttcg caccattctc 3720 ctgcctcaacccagtagctg ggactacagg cgcccgccat cacgcccggc taattttttt 3780 gtatttttagtagagatgga gtttcaccat gttagccaag aaggtctcga tctcctgacc 3840 ttgtgatccgcccccctcgg cttcccaaag tgctgggatt acaggcatga gccgttgctc 3900 ccagccaaactcgttacttt gagaacactt tttttttttt tttgagatgg agtcttgctc 3960 tgtcgcccaggctggagtgc agtggcacaa tcttggctca ctgcaacctc cgcctcctgg 4020 gttcaaatgattctcttggc tcagcctccc gagtagctgg gactacaggc gtgcacctcc 4080 atgcctggttaatttttgta tttttagtag agatggggtt tcaccatatt ggccaaggtg 4140 gtcttgaactcccgacctca cgatctgccc accttggcct cccaaagtgc tgggattaca 4200 ggcatgagccaccgcgccca gcccaataac acattttgat tttgatttta gatactcttt 4260 aatgcgttttattataaaag ttatttaatt atcattttga agtctaagga ctttcatatc 4320 tcaacaacagaccagtaaaa ttcagtgcat atttgggtat tgacaataca aatttaaagt 4380 atttattaatctattcatat ttgacaggca tctggggcca ttggtccatt tttttcatag 4440 cttcagaactaaaaagatgt cagaagatta ggtataagca aaactggtta ccagtctccc 4500 cagagctttttaaaattggg aagtgacctt cttgtaggaa ccctgccaca catgagcaac 4560 tgtccatgagcagtgatgaa tggacactga agggttagcc gcagcattgg aaacagggca 4620 gagtgagccatgcccttcct gggagctccg cttcctcttc caggacgtgc ctcttgtgct 4680 aggtaagcaccaagctggga gatggagcaa gccttgagac ttgcacttca tttagtgtca 4740 aaagcctgcacagtgtagag gtggtctctg aacagcctgc atggcaggct tttggatctc 4800 agtccaagactttctgacct gatagaaatc ccattaaaag atgcttactg ggccgaggca 4860 ggcagatcacctgaggtcag gagttcaaga ccagcctggc caacatggca aaaccctgtc 4920 tctacaaagttacaaaaatt ggtcaggcat ggtggtgcat gcctgtaatt ccagcttctt 4980 gggaggctgaggcaggagaa tcacttgaac ccaggaggca gagattgcag tgaactgaga 5040 ccgtgcactgcactccagcc tgggtgacag agcaagcact ctggcccaaa aaaaaaaaaa 5100 aaaaaaaaaattattgctta cttctgtagc tctcaggcac tgcagctaag caaggaagga 5160 attgtttcttccctcactag caggtataga aaacactgga actcttttga ttttaaaatt 5220 taaatttcagtattgacatt tctcaatagg aaatggctac tttggcaagc ggcagaaaac 5280 aaaaattactagatcctagc agggacacgc gtacaagtgt ggtagcaaag cagctagttc 5340 cgagtccctgggacatcgtc agaaggttgg ggatgctgct ggagtgcctg cactttgaag 5400 gtgtcctagatccctcaatc catatccgtc tcctcagcca gccaaaggag accagggctg 5460 ttgagctggagccctcctct ttcagcttag atccatgtgt gaataggaag gattctggca 5520 cgtccaaaaggctcatgaca tgcccagaat ggtaccatca cctaaccctg caaggggcat 5580 catttccacatctgtgtgca tgaggttcag ccccatagat agtgtggcat tgcctgactg 5640 actggagactaccttgagtc tcgctcacaa ggggaaacca tagactggaa gttctgagag 5700 cactatgtctacatgagccc cttcttgctg gcctgccttc catctcatcc ctggacacaa 5760 actgctacacaactttctta attctgactg tggatgaagg tttgtcacag agacctcagc 5820 atcctgaagttaccagaatc tttcactatt tgcagattat gtcctcatta tgagaaagac 5880 caccacttgtggtagagagt tgggaatagc attgccctgt gctcctccgg gggacttctg 5940 tgcatcctgcattatttatg agatgaaaat tatatgtgaa ggctaagtat ttcctgagaa 6000 agtctttcataccagcacca taataataat agtggtgctg ctgctgatgg tggtgatgat 6060 cacagtgatggtgatggtga tgatgatggt gatggtgatg atggtaatga ctgtgatgat 6120 attgatggtggtgatggata tggtggtggt gttgattgta atgatggtga tgttgatggt 6180 ggtgatgatgttgatgttga tggtgatgac ccttatgatg gtgatgactg atgatgatga 6240 tggtgatgatgacagtgatg atggtgatga tgatgatgat gatgatggtg atgactgtga 6300 tattgatggtggtgatggtg gatgatgacg gtgatggtgg tgatgatgtt gatggtgatg 6360 gtgatgaccctgatgatggt gatgactgtg atgatgttgg tgatggtgat ggtaaatgac 6420 tctgatgatggtggtgatgt tgatggtggt gatgatgtta atggtggtaa tggtgatgtt 6480 gatggtggtgatggtgatgg tgatgactgt gatgatgttg atggtggtaa tggtgatggt 6540 gatgatgacagtgatgatgg ttatgactgt gatgatggtg atgatgctga tggtggtgat 6600 gatgctttggtgatagtgat gatgacggtg atgactgtga tgatagtgat gatgtttatg 6660 gtggtgttagtgatggtgat gttgatgatg ttggtaatag taagaactgt tactctgagt 6720 gcctacactgttttaggtac cttcatttca ccaggacacg taacagagct ctcgtctctc 6780 tgactctgcaccccatggct atgtcctaat tccactaaac tcacactctt acaccagcca 6840 gtaaacctgttgagggaagt gctcattatt ttttttccct ggcatgcagg aagagttcga 6900 catatgtttgcaaatacagg ggtctgggac aggcggaact gcaaatgatc cttttgagac 6960 ctgaactactctgctgaatg ggcactgagg ggagatttat cgctggacag tgtagagtag 7020 ccctgcccacctacaagatc agagagaggc cctccagagg tggggtatca caaagctctg 7080 gtggctgagacctgggtgct ctgtgtgcca gtgagcacag ttttgtatcc ttcttgtaga 7140 atgggtgagattcagggagt tggtttgggg acattttact ccccacaagt gcctgccagc 7200 catgaggccctgctccctca ctctggtctg acttctgctg ccactggtgt tttgtggaag 7260 agcccctgggtgctgctgcc aggctcattg tccccgcccc actctgggtc tcagcctggt 7320 ttcagcctgcacgtggtggg ttttttgtat catacccttt taagtgggag aatatttcaa 7380 ctgatgatatcccagtaaca aaagaaatga ctcaacctgt gaaattcatg attccgtggg 7440 tggcaaaaagccagttctgt ggtttttttt cataccttga gggctctttg cttcatgttg 7500 tcaaaatattttattcatgt tacatcaacc tccaccctgt gatgacagca acctggcagg 7560 ccctgttctgcttgcagata gtgctgtggc attcccacca ggcatttaat gtccctccca 7620 gactcggcatctgctctgtt agtgctaacc catggctgat aacctgtgca tcctaaagaa 7680 agttcagagcaatggaaact gtaagctttt aataattgac catggagagc ccgttgattt 7740 aaattgtgtgctaaacccca accaaaggaa ccaaatgtga aagctggaag agcgctttag 7800 gattcagaaagcagcttatc tgcaagctct gattccgtgg cagaaggctc acagcctcac 7860 aaagtggagacaggcagaca gtcccacctc atttcaactc cagagttggg gaacgtgctg 7920 ggggtgctcagccagagcct ctcagccagg ccttgtgagg cagagggatc cttaccaggc 7980 agatggtctggaggagaggc agaccgggag aaagcatagt gtgccaggga agttcagtgc 8040 tggggagtgaggctggagat gcagataggc cccaccatgg agggccagga tgggggccaa 8100 ggagtgtggctcttccctga gggcagaggc gagccttggg gtcttgggct gggcccttgc 8160 ttgttttttttttttttttt tggcatggtt actgtcttac cagaagagaa ggcactgaga 8220 aggtgcactgagcctgggag ctctattagg gtcccagaga caatggagga tagcatttag 8280 gcaggatgggggttccatgg ggtggcattg gcctccaact ctgcagagga agaatctcag 8340 gagggagggagggaagtaag caagtgggcc ccagcgcctg cgcccgtagc agctggacat 8400 tggaagaatgaaggggcctt gaatttggct attagtgatc tgggtatggg ggtttcagat 8460 ccgtgctagggacacagggg atccatggta ctttctaaaa gtccaggtgt cgttatttcc 8520 gtactgtgtgttataactga gcagtgccca gttatgggca tcatcctgcc agtgccgggc 8580 cctcagagcatgggggaaga cagcagcgtg cgcatctgag gtggaggatg gaggagtgac 8640 cgcagcaccaggcatagaca ctcctggagc cctccaagga ctcaggcgtc agcaagagac 8700 cagggctgatggcagcacct ctgccttttg ggggctgtct ctggatggtc tcagtcagcc 8760 cagggtcacctttattctga gatgcttccc ttggccacca ccccctccag ggagctgagt 8820 gaggtcctaggacccaggtc ttataaaggg acttcctggt actcacatca gagaaccagg 8880 atagaggaggtctgggagcc aaggagccag ggccccgggc gctggacatg cgtccgtgag 8940 ggactcttgctctttgtttg ctaccactgg caaaacaagg ctttttctcc atctgtgagc 9000 ttcagtctctggaggtttct gagagagatg ggatatttat gtgccttgag aactctgaaa 9060 agagactcctgcccaggggt caggactaga cacagctcct ctccaccttc agaccccgat 9120 gctttagggccgtgggtatg aacaggtcat gtgtatgtga gtgcacattt aataaccgag 9180 tcagcccactgcactgcagg gagctctgtg ggtgtccaat tcggaaaacg ttttagaatg 9240 agaaactccagtctgcttgg ggaaactgcc attgcaatta ctttcttttt attgcataaa 9300 accaactttgaaattttaga gttgatgaag ctttccattt gccccagata ctctcactct 9360 gcaaagcagggggccttggt ttcccaccct accaccttgg tgggcgctct gaagacactg 9420 ctgagaggctagggccacag aggagaaggt gctatctggg ccttctggga ctttgaggtg 9480 tggattccagcccaccttct gctcagtcca cactgcccgt gatagctaca tcaagggacc 9540 ttcactggttcaggcccaaa atgaagtatc gtttctataa cagatacctt ctagcgccat 9600 ctttactcttctgagatgaa agtcatagcg aatataacct ccttaagcac atactgaaaa 9660 ctaaatttaacaccctaaca ctcatgtcaa aggaaaaata aaaagaaagt aattcacaat 9720 aaagtcatatgaattttgag atgaaaatct ttggtccggc ccacagtaga aaacagaaat 9780 catccctcaggtttaaaaaa tactcattaa ttttttattg tggtgaaata catgtaacat 9840 aaaatttagcattttaatca tttttaagtg tactgttcag tggtagtaag gacatttgca 9900 ttgctgtgcaaccatcatga ctatctagtt tcagaaattt gtcgtcatcc caaagggaaa 9960 ccttataccccctgaagtca cttcctgtcc acacctccct gccaaacccc tggcactcac 10020 tcatcccctcctgtccctgg ggatgtgcct actgtggaca ttttacataa atggcatcac 10080 aaagtatgtgctttttgtgg ctggctcctg tgacgtggtg tgtgatgttt tcgagccgga 10140 cgtgttacagcccatgtggg aacttcagta ctgctcctgg cagagtaata ttccacagct 10200 gggatagagcacagtttgtt tattcattcc tctctcgatg gagacttggg ttgttcccac 10260 ctttggcctcggtgaatggt gatgctgtga tcatgggtgt gcctgtgttt gtctgaacac 10320 ctgctttcagttgtttgggg cgttacccag gagaggggtt gctaggtcct gtggcacctc 10380 tgtaacttgctggggaactt ccccactgat gcttgaaagt catttggtat caccaggtct 10440 ctggggtgtttcatttgtcc ccagaagctc tgcctaagct gcactgggag tgggctgatc 10500 tgtgtgaccctaacggcctg agtgctggct caggggaact gctaatttat ggaatcctag 10560 gtaggtggtggtagaattct ctccctctgt cagggtggag cagttacgac aaatccacag 10620 tctcagggacataaagcaac atggtctttt tccaatcatg ccacatgtcc actgcattgt 10680 ggcttgacatgggcctcatg ccaggacctg ggatgagggg tgagccctct ctgtgcaccc 10740 aaggctgccgacactcccga gagcactgcc ggctcccacg gcttctgcca gaagtcaccg 10800 gctgcgtcgctccccacagt tcatcagcct ggtggacctg tggccacact tatgttcagc 10860 gcagcccatgtggccctgaa ggtggacagc ttttgtatcc gtactgaggc atgggataat 10920 aaacgccacagtgattaaaa gaggaaatgt tggccaggcg cggtggctca tgcctgtaat 10980 cccaacactttgagaggcca cggtgggtgg atcacgaggt caggagttca agactagcct 11040 ggccaatatgatgaaacctc atctctacta aaaaatacaa aaattagccg ggcatggtgg 11100 cacgtgcctgtagtcccagc tactcgggag gctgaggcag gagaatcact ggaactgggg 11160 agacagaggtttcagtgagc caagatcata ccatttcact ccagcctagg caacagagtg 11220 agactccgtcaaaaaaaaaa agatgaagtg ttagtggaag aggccagtaa ggtcacgtga 11280 tggggggtgatccaaaaaga aaaccaatgc atgtgaacag gcaggcattg aagcctgtac 11340 agaagccccattgatgaggt atttggggcg gggcagtgtg tgaggcaggc gggggtcccc 11400 cttctgtggtttgcagcttt tcacatctgc acgtaaggtt acagttgact tacaaaacat 11460 ggggtattgctggcatttct catgttggag ggcttcgttg tgtggacgta tcatttccct 11520 tttaaatatgcctttttaga agaaatgcca ccacctagcc atggacttca gtgtttccca 11580 gtggaaagaattttcaattt tgcaatctaa aaaatgggat tccccccttc cgccttctgt 11640 tgcagttttgccggcgccta cccgttctct tagaatcagg gttagacatg aagacagcac 11700 gactgagggagggtgggcct gggaggggaa ggctcttccc tgggttggtc ttgatatgtc 11760 cgtcagaaccctggctaatg atttcctaac acattacgct gtaaaaaact gcaagttgtg 11820 cggctctttaaggctatgcc ctccacgtgg aaggggctgg tcaatcagtc ctgtttcagt 11880 tttgggagaagactcctaaa accatttctg ggagcatttc agctgggagg agtgttgggg 11940 catccttgtcatgccaccct gcctcctttt acacactgtc tcgaaaagat tagtgtgtgt 12000 gtgtgaatgagctgaagtct ggaggcctca agctcaccgt gaagcagtct ttgcctgtta 12060 tttgttttcactcctaggaa tgcaagggag cttccagaac ctatcaggca cacctgtgtg 12120 atgtcgcgctttattcctag aagatcacat caagataaat cagtagctta tggggacttt 12180 accttctagtgcccagatag aaaatgaaag aaggcatgtt ttaaaatagg taatgttcta 12240 gcatgtcatgagttttcacc tgttcttatt ttttaagtta attaattaat ttatcatttg 12300 agataggatctcactctgtc acccaggctg cagtgtagtt ggtgtgatca cagctcactg 12360 catccttgacctcctgggct caattgatcc tctcacctca gcctccagag tagttaggac 12420 gacaggcatgtgccaccatg cctggatcat tttttttttt ttttgtattt ttctgtagag 12480 actgggtttcatcatgttgc ccaggctggt ctggaactcc tgggctcaaa agatccaccc 12540 tcctcagtctctcaaagtgg tgggattaca ggcgtgagcc actgtgcccg gcccctgttt 12600 tttctatgttttaattaact tatttatcat cgtatttatt taaggtgttt aacatgatat 12660 tttgtttgttatatctatac atagggagag gatcactaca gtcaaaccaa tggacatatc 12720 caccactttacctagttact tttgtgtaag agcacctaag gtctactgtc ttagtcaata 12780 ttgcatcctgaagatttgct cagggagcgg attccgtctc ttttagaaca gaggttggac 12840 actttgtcttgcatgttgga gtcacctggg gagctttaaa aatgcagacg ctaaaaatgt 12900 gggtacctgcttctacctcc agagggtggc agattgagtt ggccctggtg gggcctggat 12960 gtcagacttgctttaaaaat gctccccaaa taatgcagtg ctaattcagg gttgagacct 13020 gctgtgattcttgagcttgg ttgtgtgttg gaaacactag ggagttttta aaaatactga 13080 cacctaggtctcaccgctgg aggttctgac ttcacaggtg aggcccgagc tttaggattt 13140 tagtgataaaaggtggacaa tggcctttga gaaccactgt tgtatatttg cagttctcaa 13200 cttaggtgcgtgtcccagcc tccaggagag ctggcccttg gctcattgag gctggaacgt 13260 gccgcagggtggttctaaga tttgtgaccc tgctgtggaa tttagaggac agccaacagt 13320 cacgtattggttatcagagg cagaggcagg cgaccccatg ggctgccttc cacctgggtg 13380 ctggctttatttcaccaggg cagagtgggc tatgtgaccc cacagtggag agaatgcagt 13440 gacatgaagtgtgtgagtgg gcagcatggt gggtgtccgc tcctgaacac ttgacccatc 13500 ttccttcccctccctcatcc cacttcccag cagagcctca gggcctggga tctgccaggc 13560 cgtgcgctggcaaagatcag tctccaagag cagtctgaat cacagaatca cagctcaggg 13620 tcaacataagtactgactta cctatcagga tgtgttattt tacactctca attagtttac 13680 ataattgggagttcagtgta tttggacaag gtggaaagtt gccgaagagc tttgtgggtg 13740 ttttaaacaaaatttaaaaa acattttctg gctaagcgat ctgagaagcc ctctcctgca 13800 cttcagactgctcaggggtg aattgatggc aacagtgctg acccccaggc tgggctgagt 13860 tcagtcccacatgtgtttag tacagcaccc agtgtcttgg cctgaatccc tgattctcca 13920 gggctctgacggcgatgcat ccccttctca cgaggcccct gggtgccccc agtcaaggac 13980 tggctttcctgctctttcct cagagcacgt gaggattccc tgtcaggacc gtcccccacc 14040 ctgttgtggtggctttggat aaggtacgtc ataggatgca cctggggctg aggccccacc 14100 atccccagctcctcctgtgg ggccggaatg actgagcgtc ctcctgcctc gcaggctcgg 14160 tgtccttttgcaccctgtgg tgaatcccag atcccgctgg gcgtggcgga gtctcaggtg 14220 accctgcgcaacttgctggc tgggcagccc tcagcacgtt tccttatctg tccttgacgc 14280 cggctccacccgtgaataac agtgagactg ctaaatgagg aaacatgctg gtctttggga 14340 gtggcagtgtaggggacttt ggggcatgtg tcccatttta tactcaaggt cacgtggttc 14400 ccgaggctctgggtgggtag cacccagtgt ctggagggaa ggaggcttgg gcgaagacag 14460 ctaaatgcctggtgtgggga ctgcccaaag agatgggcag cgtccagtca tgtgaccttt 14520 ggagaccatgttaccatgtc atgttttggt ggacaatgag cgtgtcaccg cgacaagagg 14580 ggcacaagactgtgtaaaga aaaagccact gcctgtccct ccctccacag agaccccagc 14640 tgcatggagtgggtgaagct gcacatcacc tgcagatcac cggggtctgt gttcactggg 14700 ggtttcaaggggcctgagat ttctgcactg agtactcatg acctttgtaa ggagaggatg 14760 caggattttttttttttttt cctggctggg aactggcagg gaagtaaggg cagctcttcc 14820 ccagcacctgggcctcaggc tgggagcagg gtgggcacag gtgtgagaga tcagggtgtg 14880 tgtgccatgggtgcagccag atgcaggtgc ccccctgccc caccccaggg ctttgggagc 14940 tgccctttgcccgtgaacat ccctgtgtgg gagcttgggt tttgttccct ttctccttgg 15000 tttttctgagaaggggtacg ttggggagct gggggtggga gggcggatcg cccagagccc 15060 tgcaggtgctctccagtgct ggcaaattcc ctcttgtccc cttatcagct ccttccagag 15120 gtgacagccatctgtgtggt ggcagctctc tcctgctccc atgaggtctc ctttttcttc 15180 tcttttcctggatttgactt cttaactgaa cagaaaaata tataggcatg gacagttttg 15240 tagtagtgtttggagaatag gccttaacaa tataattatt caattcatga tttcattgtt 15300 caaactgcccaatctcattg cattacggcc agttctttgt cgtaccttgg aatttgtgtg 15360 tgtgtgtgtgtgtgtgtgtg tgtgtgtgtg tgtgttttgt cctctgtgtt tgtttgtttg 15420 tttccttttgatgcatataa tgtaatattc ttggtgaagg ccatacctcc cctttctgca 15480 ggtgtgagtgggagggctgc aggttgcgga gtgagacctg aggctacact gctggcccct 15540 gcagttttaccagaagctca aggataggat ggttgtaggg tgaggcccct tgtggtccca 15600 agcaagaccagggcagctga ggagggtgga ggaggagggg tgctgggctg tggtaggagg 15660 gaggctaggggacaggaaga gactgcttca ggtgctggtc atttcacatt gatgtgaagt 15720 gtgtaagaagtaattgatgg actcaaacaa ttaaatgatt ccctcgggga gtgctcagga 15780 attaagcctcagtgaggaaa aagaaatgta aaagtcatgt gtgtcttcat tgagatatgt 15840 ggctcttcacaggttccagg gagcttggtt tgcttccata tgtttctatg tgtttttcta 15900 atcttctgcaatgatagaac atcatactaa agaaaaccat aaatcagtca ctttggaaga 15960 gagatttggggtagagagag agtgttttac caagtgctag ggcctccttt gggtttaatt 16020 cagaagtcagtgcctcagcg ccagggctcc taacctgtgg cctgggctca cctgttcacc 16080 cctcacctcttcaccccttc agagctcacc gacttagcaa agaagtactg aacccgcagg 16140 gtgtctcagcttggacagac tttggagctg aataagacct gatccttgtc cccacctggg 16200 acagctggctgctactgcct ctgtcctcag aaagcaatcc aagagctctc tgagctggca 16260 gtgcagaggtacggagcttg ctgtttatgc agaccttctc tggagaggtg agcattgatc 16320 ctggtaggcagcgtaccaga gctagcaaat gaaccgcctg aggaaatgtc cccaagctgt 16380 ggcctctcataggtcaccca gacatcagag tgtgaagcaa aagtgctttg cttctcccac 16440 ctctgccccacctgtagcca catgaaaaga gtggttctgg aagtccctgg tgtaactttg 16500 gtgtagcacatgggctgtgg gctctgtgtg cacactcgag acccagcgca agtcaccact 16560 gccatggagtcacctgtgga cccaggggct gggctagtca gagcagtaga tgggatgagg 16620 atctgggggccttgagtgga ggggcagctc tcagtgagaa gagggtgcct ttggctgcat 16680 ctgcccctggaagggagcca ggaaccaacc cctctacagt ggagatgctg atgggtggcc 16740 tcgtcctggagaaggggcca ggatggaaag gacggacctc caggaacagc cagcgccctg 16800 gcttcctgacaggcagctcc caggtgggga gaagggcttg tttgacacag gtgtgaggtt 16860 tgcagatacagggtgatgag gtggggccag tattcagaat gcagaacctg accctccacc 16920 atcacggaccatccgtttgt gagcagcaca tgcagctgtg gggccgtgtt ggagcacagg 16980 gtgggtgtgagtgagcgtgt gagactgtgc gtgagtgtga gtgtgagaat gagtgtgaga 17040 ctgtgtgaggctgtctgtga gtgtacaaga ctgagactga atgtgagtgt gacaatgtgt 17100 gtatcatgaaaaacactgtg agagactgca cgtgtgtgag tgccagtgag tatagtgtaa 17160 gtatatgagaaagtgtgaga gcatgagtgt gtgtgtctgc ttgtgactgt tgtgtgaatg 17220 tgggtgagagtgttagtgca agggtgtgtg agtgtaagag tgtgtgagag actaagagtg 17280 tgaagtatgcaggtgtgtgt gacgtgggtg taagagtgtg tgtgcaaatg catgagtggc 17340 tgagtctgcatgagtgttat gagaaaatgt gtgtaagaga ccacgtgaga gtgagtgttc 17400 atacatgtgtctgcaagtgt gtgggtgtga aaaagtgccg gagactggga gcacatgaga 17460 gtttgactgtgtgcatgtga gtatgtgggt gtgttcctgt gagagaggtg cggggtgtgt 17520 gggaggggcagctccagtct ccagggagca ctcctagcgt ggtccattct tcacggacag 17580 gagcccgttttgtaacccca ctctgagctt agttcctgct cgctgttgca cccgtgccct 17640 agggccttccttggttcttt cttgcacaca gcagacatag gtgccgtggc ctggcttcca 17700 ttgccatctctgccctagac gttgtgcacg ggttaaaggg aggcagcgcc tggtggaacg 17760 tcctgggcttggatgtggca ccggagcctt gactggattc tgttccacta ctgggcttcc 17820 tgggcatctaccttggacct agcgtggagt ctcacgttca agttgtcttg cctgaagaag 17880 acagctagtgggcctgcctt gggtctctgt tgaacaatta aaggagcacc gagatcccct 17940 ctgttgaataattaaagaag ggagagagag acccctgctg caggacgcct tgcattgtaa 18000 cctctgggaagcataagccc tgctcgctat aaatgagaat gtcacttcca catctcctct 18060 gagcatattggtccagaaaa ctacctccca cattaatttt catatggcta tcagacatgg 18120 gagatttatcagagagaagg aaaagtgtaa ggatcaaaca cgtgagtttg ggagaataca 18180 ggaactaatactaatgcaaa attagaacgg gatttaggga aattcagtac agcagcacag 18240 agtactacaaaataaaagaa gctttgttcc tctcgaaggt aatctgtgtg ttgtttccct 18300 aattttaaaaagtaatacat attactggcg aaaaccctga aagatagaaa catttccgaa 18360 aaaatatgaatggtgcctag aattacacaa cccagagaga agctcttatg agcattttag 18420 gacattttcttagtttttaa aataaaaatg aaaccacact acacatgcat ctttgcccac 18480 tgtcttttgtcacttaatgt tggagcgtgt ttgtccactt caagttccat aaacttgact 18540 tttaatgacaacatgaacag cccattttga gtgtggccca ttataacact tactgcctgc 18600 aactgtgtgccaagccccag cctttgctgc attatctgta ggatccccta agccagcttt 18660 gtgttgttcttatttcattc tggagatggc tttgaggact cgtcccctgt gagcagagag 18720 gcagagtccacccctgccaa gtggcccctg ggctggcctc tgagtgagca tctactgctg 18780 gtgacaggacatgggttcaa cagcagctcc cgccagcccg gtagcaacgc cttgagcaag 18840 ctgccctctctcttcttctc tgaccttccc cgtagccaag gactctcaga tgggaagaac 18900 ttgaaaactgtccctccttc cttttctcta gactgctgtg cactcagacc ctttggaaat 18960 gcacccccaccaggttctct gctggacccc atgtatttac ttagacactt ggcagatttc 19020 ctaagactgggaagaataaa ccattgttaa atctgaaatt cacacgagag acagaagaat 19080 ttgtgaagcctttgctggct gccccatcag gacatgtccg ggaggaagag tgccccttcc 19140 tcagcaggatgagggtagcc atcttgttcc caggggcccc tctgtcacca caggcagtgg 19200 aagtgggcagtgcctcccag ggctcatggg gcccctgtct gccccgtgta ggctttttgg 19260 ggtgggaagggatggaaggg aaggaaaggc accagtgggt aaggaagagt gaccacagag 19320 gaatagagttggcagttgtg gattttaaag tcagatacat gtggtgacaa atcattgctc 19380 tctgctgacatgctgtgtgg tgtaggatga gtactcagac ctgagcctcc attctccttg 19440 tctgcaaaaaagacgtagca ggccgcctta atggagtgat aatgggcttt tctctatgag 19500 agcacctgcccaggcttaga ctgtcactgt tgctggataa acgttggtct cctgtttttc 19560 tttcttttgtaactacctcc tgggcagatg tgggtgcatc ctagcacgta gtggttttgc 19620 agtatcttaaaccctgtctc attttgtcct gttttttcct ggcgtttcaa acttctcatt 19680 tccaaggtttgcagtccgag aggaagggat ctccacaccc cacagaggcc tgagccttgc 19740 tgaccttgtgtgcctgtgtc ttttcccttt tgggaatgga aagcaggacc ctgcagggct 19800 gaagccacactttctgtaca ggccacatcc tggaatccag ggcccggtgg ccctgctttt 19860 gcaggtgccttgactccccc attcttcctg cgcttgcaga gcctctttct ggcctcagac 19920 ccacagctgctctccgtcgc tgagttccct ccattgtgga aaccatctta ctcctggtgg 19980 tcccagtggcaggatactga gaagtattga tccatccaga gggagaggca tgtagttctg 20040 cttaaccagacatggtgcca cacagatttg agcacaagca ccggaggtgg gacagtaaga 20100 acattttttttgagactttt tttaaatgaa gattgatttt ttttgagact ttttttaaat 20160 gaagattgatttttttttga gactcttttt gaatgaagaa cattgttttt cgttgtaatt 20220 ctttggggagagagagaatt gaggaacagt ttcaaatcca tgtgttcaac aggaacattt 20280 ctggggccagctcacagaat gcctgtgttt cagagcctcc tctggtgtgg gaggcccaga 20340 acggagcccactgttggctc tgtgtcagct catgcacaga ttgtattccc tagctgctct 20400 aaaggagacatgttgaagga aacttccaag ttgaaaggtt agaagaagtc taagcgtcct 20460 gcattgagacttatgtttta gtcctggaaa ttccttttcg gaccctgtta cgacctggtt 20520 gtgccttcgagtgcagctgc aaggctgtgc tggctgttgt cagctaaccc taatgggacc 20580 tcagtttctgaagccagtga gtttccaacc tcaggcctga gatcagagtg cgttaacatg 20640 cacttcccatcctccttctc cagatcaaag agaacacttg accacactgt ccacagtttt 20700 attattattactatttttag ttgagacata ataattgtac atatttatgg ggcacagtgt 20760 gatattttgataaatgtata caatgtacaa tgctcaaatc ggtgatttgc atagccattt 20820 tttctgtgttcttcatgcaa cactgcatgt tggaatgata ctgccatgtc atttttgata 20880 aaacaccacatattatcttg catgttgaac agaagtacta ttccctcccc tttgaaaatt 20940 acctctctagaagtgacctc tgaagttcta ggcagtgagt ggactgtaat gtcagtacag 21000 ggcagcggagctcctggtgc tctccaaaga taggtcctcc ctggtggaat gatttccctt 21060 gaggccagctgcatggctcc tttttccagg aaggggacca ggctctcagt cacttgtctg 21120 tggccatgacacttcctttc cgtcactaac acccagggct atcgcctgcc accttgactg 21180 cttcaggaacaaactccacg tctgagctga gtaccaggtg gtgcgtgggg ccagcccaac 21240 tttgtgacctcacacctctg ctagccctct ttaaactcat tcttggaatg gctgcaaatg 21300 catgtgtgtgttgaagagca ctggagtgag ggtggtcccc tccctggcct gcagcacttg 21360 cgtcctgtggagggtggtga gtgtgaggat gaggaccact gcaccatggg gatgaggatg 21420 ctgtagtgaggggctgctgt gtgagtagga ctctcagata tgccatttca tttcatttaa 21480 tcctcccccttgtgggaggg aaaagagagc aagcattatg ctcattttac ccataaagaa 21540 atgggggctgaggatgtcct gcagctggtg agggatacac gtgagacccc aaatcccagg 21600 tcctaacgcacacttcttcc tccttctttc cctggatctg tggagggagc tgctgggaac 21660 cgaagagaggatggaaggaa gaggtggaag gttgcttgga aggacctgtg tttggatctc 21720 ccttgcttggtcgctgttgt gtggtgctga gcagatgtcc catgggacag ggagggtggt 21780 gtacagcctggctcagccca ggagcctcct taagcctcac ctgaatggca tctagtagct 21840 ggtagggtcgggcagtggag cccccccatg ctgcagacag aaacaagaaa ccttacagcg 21900 gtcctgctgctcctctcagg caggcatggt gggttgatgt cttgtcgctc tctgtggaac 21960 cttctctttatcatgccgac ctttgcattc ctgtgagctt ctgaagtctc tggatgtagg 22020 aggtgtcagtggcatatcat ggactcagca cgactgtatg tcactatcat gatgtatttt 22080 ggagggtggccatcaaccaa tttggaacaa agtcattcta agaggagcct tatgtgggcc 22140 actcatgtttatgaatcaaa ctccatcttt cagtgaaatg ctactctgca ctctacactt 22200 acagggactatgtatttata gtttaaatct aaactgtagg ggacagtgtt gttgggtttg 22260 catattcagactatgtcatc aatagaaaca ttcctctttt aatagccggc agcaggtttt 22320 tcctaactgccaaatgataa gaatccctgg gaagaggttt tagaaaatta gatcccagcc 22380 cgacccccattttcaccaac acaaaaagaa cagaatttgg tcaagagtgg ggaccccagg 22440 gatctgtgtcaggaataacc gcctgtggtg tttctggcaa ggaaagattg ggatggtgaa 22500 tatttattgttgatgcttga aataataggt tttttccctc tccagaattt ctcattgcta 22560 agattcaaattatcatagtc gggccatgct ctgcccaagg tcaccgcagg tgtgtggtcc 22620 tcagggcactctggtcctga caggacacca ggggtggcat gtgggggcct ccttcccagc 22680 tgggtcatcgtcagtcccac ccagcattcc atccgggcac tgtcctttcc cagcactgag 22740 agtgagtctgggtgcttggt cctatctcca agtggcctgt tctggaagga gcatgctggc 22800 ctggggaagctgcacatttc caaggctgtg gccttgttca tccctgagag tctgagaatg 22860 ccctgggtttcccatcagct gccttcagca accacagcat cttcccagag cagtggtgac 22920 caggatgcagcatacagcac ctttgagaag cagcaggcaa gatttaagga aggggcgcct 22980 cactccgccttgaggaagcg ccctgcagcc tgcttgtctc ccttttaaca ggctcctctg 23040 ctgaacagcccctgggcggg agactcagga agctgagcct ggggcagtac gacaacgatg 23100 ctggggggcagctgcccttc tccaaatgtg catggggaaa ggctggtgtg gactatgccc 23160 caaacctgccgccattcccc tcaccagcgg acgtcaaaga ggtatgtgcc acagccaggt 23220 cccagtttctgggcccattc cactcccttt cttgtcctgg aaccccgagt gaatgggcaa 23280 tgaccaggccttgtccctga atacagccca cattatcctc cgcacccacc ttccgtcctg 23340 gtttcacaaatatggtcgtg gctcctctgt acctggcaag cggtgtggct gggagagcac 23400 agtgtgcagtcagctggcct gggtaatgca ccccagagat agctatgctg gtgaggccac 23460 ctcactcagggcactcgagc ctctatgttc taatctgtag aatgggcaca aggagcacag 23520 catgtgggtgggttgtgtgg gttcgaggta gggctcttat aggcctggcc ttagccattc 23580 tccatagcccgacagcagca gccagtggca cacacagcct tccttcaggg tcatcacact 23640 cacacagggtcctcagtcct ccctccgaga cctccctagg aggtgaccca cagatgaggg 23700 ccacagatgagagcttcttg ctcttccaag aggcctaggt aaggcttcta gtcagaatgg 23760 atgtaaagcaggtcggtgta tccaaaacga ttaattattt tagacttggg tgggatttct 23820 tttcaagttggcaaagttgg aagctacaga attcacaaga aaaaaaagct attcatagtc 23880 ttgactcacaaaggaaacca ctgttaatat ttagaatatt tccacccggc cttttttctg 23940 tgctatgtatttaaaatact atgtgttttt atgcatctgt gaatatatta ataccatggg 24000 taaggactgttttatccagc tttttcgctt aatattgaga tactcattta cctgtaccat 24060 tacaaactctgtctacttca cttattagct gtatattgta ttagaaggat gggccgtaag 24120 ataccttccgtttcctttac tgttgaaggt gaaagtgttt tccagttttc catggctagg 24180 agaggcgggctggttttgtg cagtggatcc agacctcagc ctccttgtcc tactgtcaga 24240 ggaggggtttgctcagtggg ccagcctggg ggagccacaa ccacaactgt ttggaggggc 24300 cttcacttgactccctgttc tcacattcta ctccctgtgc caatttcaga cgatgacccc 24360 tggctatccccaggacctcg atattatcga tggcagaatt ttaagtagca aggagtccat 24420 gtgttcaactccagcatttc ctgtgtctcc agagacaccg tatggtaaga atgaagtaac 24480 actagctgcatgtgctgctc catgcccaag gagggtgaca acagcccctt gctctgcttt 24540 cttttccagtgaaaacagcg ctgcgccatc ctccgttcag cccacctgag cccccgctga 24600 gcagcccagccagtcagcac aaaggaggac gtggtaagaa atctacccgc gggtcacagg 24660 cgcgcggcactcaggcgctg catcgcgggg tcgccatttt tacagagaag aactcgtatg 24720 cagaaatgtttccatttgtg tcgtatctca gaagccttgg atgaggatgg atcttgctgg 24780 gaaagatgtagggatgtggc cgtgaaatcc actgtgtggt ggcaacctgg ccttgcatcg 24840 ctgcctggcatcctgtgggg aaccctcctg taatatgcag ttgatgggac catcagtggt 24900 gactgtcgggcagcttagca ttggtgcttt tgaagaaacg agaatgccca ccccacactt 24960 cacgttctctgtggggattg tgccgcacgg gcgccccagc tctgccaccc cagctgcctt 25020 ctcacacgtaccttgtggcc cctcctgtct gttattagtg ggtgcctaat cactgtccct 25080 ggatctctgcaggaactcag gggctctgtc catggcctga tcgcgtccgt ctgtctgcat 25140 agctggctcaggcgagagag tgttagtggg cggtgtttag ggcgagttac tgtgcacttc 25200 ttctgggtggacttgaggac ttgcctgaga gtccaggaca aggccgagct caggcaggtg 25260 gagagagaatgtaaacctaa agctcgtgca catttcaaaa tcactcttct ctgggagttt 25320 cgttcattacgctgctgctt ttttgcccta gtgaaggctg ctgttaatgg ttattaggca 25380 cgttacctgagactgagact ttcattaact tgtattttca tctacccctt acccaggagt 25440 gttgtgatcatgacagctag ctccattctc tctctttctc catagtaaac caagcctgtg 25500 gactatgtaatagaaagaca tccttctcct aaaattccct ttatggtttc aggaccataa 25560 attgctctcttcatatgtag catatagcag ttatactgcc tgtatttctc agtaagtgct 25620 actgaaatttaaaataagct gcattttaat gtctacatta tgtgagcatt acatgagaag 25680 tgtgtatgttccaatatgca ttagcatgat ggactcacac ataggtagcc atgcatctgt 25740 tctatattttttatcatttc aatagatgca gaaagaggga tgtcccttcc accccatgta 25800 acccctcctcctcatggagg tgactgtact tggctcctag cttgaaagtg ttcagtgggt 25860 ttgggctggcatctagccct ctggcctgga atcctgtctt ctcctgcaac accaaaaccg 25920 ggtttgtttgaaataggtag atgacaaaca gtggaaggtg ggtaataagc ctgaggaaat 25980 aaaatactagaaacagcctg caaactcatt aacagctcct gagggaggcc tctaagatca 26040 ctgtattgagcataagagaa tgtggagccc cgtctaaact ccagaagggt ctaggaggct 26100 gcaggaagcctgggtgctca gcgtggggta cccagtggcc tggctgtctt atgctggccc 26160 gattgtcccagcctggagat gagctccatg tcctgtgcgt ggatgagcgc tgcactatgt 26220 gtggatgaggatggctctgc acaccagcat gtgttagatt ccagtggctt caacttgtcc 26280 tgagtactatctgcatgtac atattcacac agaagaagtc agggctttgc ctttaaatat 26340 aaaaaggtgacatgataaca atattaatgt gagccttcta gatttttatt tcagcagtcc 26400 ccagggagggtcaacttcac attagcccac aaggagcaca gagataacac agtgctgact 26460 tagtggatgctttcatcaga cagtgtggtg agccagtgcc cttgccagga ggccccaccc 26520 actctgcaaagtcaagtgga gttgtgagac tcctccaagg gcacagtaat aacagaggta 26580 gctcgcccatacagaatgtg tcccctgatc tttatgattt ccttccattt cacagtcaca 26640 ttctatgggtcactcttact gttaccccca ttttacaggc aaggaaattg acacgaagaa 26700 gttgagtagctagtaagtgg caaagtcaga gtttcagctc catcagccga ctccacagcc 26760 atgggcagattatacagggc tgatgtttca ctttgtcttg tctaacctag aagtgaccta 26820 atgtcacaactaaaaagcac atggaaagtc tcacagtcat ggaataacca tgtaatttgt 26880 acatttaggtaacccctatg aacctccacc tccttatcta aaaatgcaga ctatagtacc 26940 tgccattcacagccactgtg cagttttgaa gtagtaggct ttaggcatct gcagtaatgc 27000 ttaactgcgtatcatttagg cctagatact gatgtgcata taaatgacag tatttgcttt 27060 aaaataccattagggagagg cttcacttcc cggagggtga attagacata cttttctcta 27120 tttctcctgctaagtacaac ttaaaacctt ggacagtaag tataagacaa atataagaaa 27180 gctctgaaagatggggagaa ataggccagc tagccatgga cttcagggcc aagggacccg 27240 gtaggccttatttataatat ctatgggcca ggaaacaatc ttcagaggac taaaagtaac 27300 tgaaccatgcagaacatgtt ctccaaccac aatggactca ctagaaatca gtaacagaag 27360 ggcaacaggaacaccttcaa cacttggaaa ctgaacagca cacttataaa taatctatgg 27420 gtcaaagaagaggtctcgag ggaaatccaa aaacatactg aactgaatga aaatgaggat 27480 aaaatatcaaaaattgtgag acatggcttt atccactgct ttaatacagc cgcactgaga 27540 ggaaattttgggcaagaaga aaattctgaa atgaatagtt tatgctctta cctccataac 27600 ctagaaaaaggagaacaaac tagacccaag gcaagcagaa ggaaggagat aataaaaatc 27660 taagcagaaatttaaaaaga tacagagaag taatagagaa aataaatgaa agaagtggtt 27720 ctttgaaaagatcaataaaa ttgacaaacc tctagcaaga ctaacaaaaa aatggaggac 27780 atagattactattgttagga aggaaacagg caccatcact aaagaccctg cagacaccaa 27840 aagaataatgaaggaataca caaactactc tacatgcata gtttttaaaa cttaggttaa 27900 atagaccagttccttaatga gcatgaactg tcacaactca cccaatagga aatagactac 27960 ttgaataccctgtaactata aggaaattga cctagtaatt tataatctcc caaaaaagaa 28020 atcttcaggtttcactggat aattctacca aacatgtaaa gaagaattaa cactgattct 28080 acgcaataccttccagaaaa cagaagaggg gggatcatgt cctaattcat tttgtgaagc 28140 tagtattaccctgataccaa aaccaggcaa aaagaacaga aaaagaaaac tacagaccaa 28200 tatccctcgcagattcagat acaaaactcc ttaacaaaac ataaacaaat agaacacagc 28260 aatatataaaaagattgaca cactgtagcc aaatgggaca tattctgaga tgcaaggctg 28320 gtttagtatttgaaaattca ttcatgtaat acactatact aacaggctaa agaaaaagct 28380 cacgactatatcagtcaatc caaaaaaaga ataaaaagag tttggcaaaa ttcacatcat 28440 tcatgactcaaaacttccag caaaatagaa gacaggtact ccttcaacct tacaaaaagc 28500 atctaaaaagccccacagct gacagcactc tttttcaaca tagttctgga agttctacag 28560 agacataatacaagaaaagg aaattaagac aacagatgag aggaaagaaa taaaactgtc 28620 cctttttgctggtgacatga tcatctacgt agaaagcccc agggaatctt gagtctgctg 28680 ctgacatgattgtccaaaca gaaaatccca gggaagaggc ggagcttgca gtgagctgag 28740 attgcaccactgcactccag cctgggcgac agtgtgagac tctgtctcaa aaaaaaaaaa 28800 aagaaaaaaaagaaaatccc agggaacaac aaaacaacaa caaaaagtga aactgttaga 28860 acaaattcagccactttgca ggattctagt tcgacacaca aaaactagtt ctatttcagt 28920 acgctagcaatgcacctgta gaagctgaaa ttaaaaatat aagtcacagc ccggcggagt 28980 agctcacgcctgtaatccca gcactttggg aggcaggggt gggcagatca cgaggtcaag 29040 agatcgagaccatcctggcc aacatggtga aactctgtct gtactcaaaa tacaaaaatt 29100 agttgggcgtggcagtgcgt gcctgtaatc ccggctattc aggaggctga ggcaggagaa 29160 tcacttgaacccaggaggtg gaggttacag tgagctgtga ttgcaccact gcattccagc 29220 ctggcgacgagctagactcc gtctcaaaaa aaaaaaaaaa aaaaaaaaaa aatatatata 29280 tatatatatatatatgtata tgcacacaca catatattcg tatgtatata tatacacaca 29340 tatacacatatgtatacata tatttatgaa aaggacatag aggtggcata tgatcgcata 29400 tgtatacatatatatatata tatatatata tgccacttat aatagctcaa aaaaattaaa 29460 tggttagatgtaaacctagc aaaccatgca caagacttct gtgctgaaaa ctgcacaatg 29520 ctgatgaaggaaaccacaga atatttcaat aagtggggag acatactgtt ttcatggatg 29580 gaaaactcaacctagtagag ctgtcacttc tttccagatt gacagacagt tttaccacaa 29640 ttcctatcaaaatctcagca agattttttt gtagatatag acaacataat tctaaaattt 29700 atgtggaaaggctaaggaac cagaatagcc aaaacaactt tgagaaagaa ttaagtggaa 29760 ggaatgaggttacctaattt caagacttat tgtatagcta cagtcatcaa gactgtggtg 29820 tggatggaggaacagacact taggttcatg aaacagagta gagaacccag aaacaggcct 29880 acacagatatgcccagctga tttttttgac aaaggtacag aagcaagtcg gggaaggtca 29940 gcctttcaacaaatgatgcg ggggcacctg ggcacccaca ggcaaaacaa tgaacagcca 30000 ccaaaggctcacactttata caaaaattaa cttaaaatgg atgatggact taaatgtaaa 30060 atgtaaaacatagtattttt taaaatggga gaaaatcttt gggatcacta ggcaaagagt 30120 tatgaggttaggacaaaagc atgaaccata aaaggaaaag ttgacaaatt agactttata 30180 aaaattaaacatttttgcta tggaaagaac ctgtgaagag gataaaaaag acaaactgca 30240 gagtaggagaaaaggtttgc aaaccacatg tccaaaaaaa ggaatatcat ctagaatgta 30300 tataatctctcaatactcag cagtaaagaa caaacaattc acttagaaaa tgagccaaac 30360 tattattaaaaagtcgaaaa acaacagatg ctggcaaagc tgcagagaaa aagaacatgt 30420 atacactgttggagggaatg tgaattagtt aagccactgt ggaaagtagt ttaaagattt 30480 ctcaaagaactgaaaacaga actgccattt gacccagcag tcccattacc ggatatacac 30540 ccaaaagaaaaaaaatcatt ctaccaaaaa gacgcatgca cttgtatgtt tatcacagca 30600 caattcacaatagcaaagac ctggactcaa cctaggtgcc catcaacagt gaattggata 30660 aagaaaatgtgggacatata caccatcgaa gtactccgca gacgtaaaaa aagaaccaaa 30720 tcatgtcctttgcaacaaca tggatgcagc tgtaggccat tgtcctaagc aaattcagta 30780 tcccctgaatttaaaataaa agttgaaatt aaaaagaaaa gccactgact aaattaaata 30840 aatggatttttattcagtag gaaaaaaaat tagaaaatgg ttcaaagaaa aaaagagaca 30900 tgtcactgaagaggacgtag aggtggctta tgagcacgtg aaaagatgct caacatcatt 30960 agccattagcaaaatataaa tttaaatcac acctatcaga atggctaaaa taaaaaatag 31020 tgacaacatccaatactggt gagcatgtgg aaaaactaga ccccttatac gttgctggag 31080 ggattgttaaaatggtacag ctgctgtgga aaacaatttt ggcaatttct taaaaatgaa 31140 aaacctacatctgccatgtg actcagtgat tgcattcctg ggcatttatc ccaaagaaat 31200 agacttatgttcacataaaa cctgtacagg aactcttttt actttaatag ccccaaactg 31260 tagatgtccttcaacaggcc agtggttaaa caaattgtag tacctcaata ccatggaaca 31320 cttctcagcaataaaaataa gtgaattatt catacacaca acaacccgga tgggtctcta 31380 gaacattaggttgagtgaga aaagtcaaaa cctgaagtct gtatagtgta tgattccata 31440 tatgtcacactttttacatg gcaaaattgt agacacagag aagagattca tgtctgctaa 31500 tggttgggaggggcaagagt agaaggggaa tggagtagcc aaaaaggcaa caggagattg 31560 gcagaagagtggtggagtaa gaggtcggga aggtggagcc aggccaggag ccgttctctg 31620 tagttcccagaaggtggtgg tgtggcatcc acatgcaatg tgaggtcatt actgaacctc 31680 aggatggggaggtgactgct cagatttgca ttttcacaca tcactgtggc tgctgcatga 31740 agaacacggtgggatggaca gtgggctgga acagaccaaa tggtttcaga agagatagga 31800 ggaaatcagaacatttgtgt aggattcagg tggtgcaatc agctgacctt aacacagtgg 31860 aattggagcctgactgctct ttctaattgt attgcttttg tcattattgt ttaaattagg 31920 ttctggcttcttgttaatta gtatgcagtt agtataacac tatctccata aaattcctct 31980 gcagatcgcattactaaaac ataacttgat acctccttag tcagcataag ccccatgtgt 32040 taagttcttttccagacaac caccaggata ggtgtccaag agaaatagcc cagttagtta 32100 aaggctgtaaggacagattt tattcaggat tattgcaaca ggagaaagtg ctgggctcag 32160 ccccaaatgcagcaaagaca gctggagatt tatgaccgag gaatagagca aaaggatctg 32220 gtggatggaaagtcattaag aagggacatc aagggtaggg gggttcttgc taaactggcc 32280 taacacggtttctgctacag acaggccagg gacttagaca caaatggcga gggatgagga 32340 gcttgatgacgtgtcaaggg ggattagata tcaagtgtgg gggatgaaaa gcaggattct 32400 tgctggaactgagcttgcca cggttggacg tggaaggcca atgttgaggc ctagtggaga 32460 agagggcttggaggagcctg gctaaggttt ggtcaaggag agtctgtcac aagatagacc 32520 catctgtatccaggctaacg caatagggtg accaaatgca gcccgcacgc ttctctgtgt 32580 ttctctccaggtcctttggg cacttagttt ataatttttt attttctatt ttttattttt 32640 atgggtacatagtataggtg tgtatggagt acatgagatg ttttgataca ggcatgcaat 32700 gtgtaataatcacatcatgg aaaacagggt attatgggca cttagtttag ccataaggct 32760 tccgtgccaagtggcctggg agccagctgg ggccagcttc tgccacaggc atccttcacc 32820 cacccttctcacaaacaagc gattcttcaa taaacaataa acagaataca caaaaagcaa 32880 accaaaacatctctagattt attaatagat gggtgaatta aaggagtctt ttccaaataa 32940 tgtgactaacagaagccaag ggggatgaat gcccaaagct ggggcttgcc agaacctgct 33000 ggtaccgagagctgattgct gaagtgtgag gaattttgtg agccagctga cagcacattg 33060 gcagcttgggatctgccagg gcgggattgt ttacaccaca ggatttagca aacgttatga 33120 atcaacccctccatcggccc caagcttccc tgacacacca aacagtagaa attcactgcc 33180 ttttttttttttttttttaa ctctgcttgg tgttagctaa ctagtcatga agaatagagg 33240 ctgaagtgaccaaagagatc tttctgcatt ttgacattca aatatttgga aagattttcc 33300 gcattcagcatttggattct agtcccattg ccaggctcaa gtaagctctg tgataacaac 33360 actcagctgttggaaactgc tgtccgaagc atctctcagc agatagggag gcctaaaagg 33420 acacggcagagtggtgtctg gtgggtcctc tcctgacatt tcttctctct ctggtcaaat 33480 gctggatgcacacatatagt ggctctgaga cttcctatct gggtgagctc aggcagttct 33540 agggacctctctgagcccta gtttcatgtc ctgtacaaca gtagtcattc ctactgcaca 33600 tggttcttgagaagattgga ccagaacatg tagctctggc tcctggccca gacctgatca 33660 acactcagcaaatagtggac actgatgctg ttaggagaac agagcatcca tatgaagggc 33720 ggttccctgagctggggaga ccagtgacaa ctgtaagtct gggttcagag catgagtcgg 33780 gggacggagaaggcccctgg gcatctggcc ctgggtggct gagccctggg aatcctgctt 33840 ttcccagagatgtgcttgtg cccagtgtgc tcatgtttta aatgccagca cctactccag 33900 gaaaatgatgggtatttcaa tatctgctca cattatgcat atttaaatat gaatttaaat 33960 actgtttaattctcctaggg atgtgttgtc atttaaaaga ggttgtgatt agcatgaagg 34020 actggggacaggagcaggag atggcagagc agctgtaggc actgatgtaa tgcctccagc 34080 tttatgaggacgggcccaac caggcttcac ccatttgggg ccctcaagcg ggagcgtcag 34140 tggcagccctcgccatcgtc cgtctgttga atgtattgcc acatggccac attatgcccc 34200 aggagccatgtgttggagga cagtatcctt aggcttctca gaaattccac atgggagcct 34260 caaatggacatgcttttctt gaaaacaaaa atatttgtaa aaatgtaaca atgggagtcg 34320 tttaatctcaagtctcccct ataagcaaaa atagataaaa ttatgaaatg taagatgtaa 34380 attattctgtttcttagctg atccttaatc ttgatgatat tttcaaaaca aatgaataat 34440 aaagacatgagaaaaggttt cctttaaaaa gtcgtggttc tcgccaggcg cagtggctca 34500 tgcctgtaatcccagcacct taggaggcca aggcaggcgg atcatgaggt caagagatag 34560 agaccatcctggccaacatg gtgaaaccct gtctctacta aaaatgcaag aattagctgg 34620 gcatggtggcgcatgcctgt agttccagct actcgggagg ctgaggcagg atttaaatac 34680 tgtttgaacccaggaggcgg aggttgcatg agccgagatc gcaccactgt actgcagcct 34740 gacaacagagtgagactctg tcaaaaaaaa aaaaaaaaaa aagagtcgtg gttctgatac 34800 atctcagcttccaaactatt tcataatttt ttaaaaatat aaacacataa cataaaagca 34860 tctatttgaaggtgaacatt gcaccagcat tcagtagatt tacggagtca tgagaccatc 34920 gcccctaggtagttccagag catccttatc ctataacagc tttacaagtg gtattcagag 34980 ggggtttggggactttaggc ttcagtgaaa aagattagca gttaatgtta gtttacttca 35040 agaggatgcctttatatatt ttgcaacttc tattttgtgt tcagttcttc tggagagtag 35100 ggtgtaggcattttaggggc ctggcttcca agcatgcagc ctcttctctg ctgtcgattg 35160 gaggttttgtgaagggctgg gaggattgcc tccatccaag tgccagtgca tgagactgat 35220 caggagtggtagcatgtgaa gtggtcctca ttggctcaca agaggggaca caggggtttg 35280 gttggtgctgtgcggacttc ctggatttgg taggtttcaa agggtcatga tgtttgccac 35340 agggaggaagaaaagggcag taccagtggc actggtcctt accctctgta gctctatgat 35400 cttgaacagtttgcataacc tctggaagca tcatttaaat ctaagcaaaa taagaatgtg 35460 tgtgccatgcatggtattgg gggcagtctt aaaaatgaag atttcagcct atgaatatgc 35520 tagtgtaacaaaaatttgtc ttaaaattta gttttttaat tcagaatgca ctttcttatt 35580 gaaatagtttcagagcccca gggactgata gagagacaca gcgtgagaag aggacaagag 35640 gaagacagagacagagagat tattttgaca caatgcttta tttttattga aaaagctgtc 35700 atccttcaaaaattgacaga tacaatgctt ttaccaatct tcttctttct ttcctgtact 35760 cgactcccttttctgttgaa tttttatgag ctctgaatga gaatgtttat aatagggaca 35820 aaggcatgtactgaatgtga atatgtgtga catttataga ggcccttgtt tactttggat 35880 tttctagagcctcaaaagca catgcattcc tatatggatt tctccatggg tttatattca 35940 ggtacgtaggcatatgtaag tgtgcatagg aatagacatg cctggataca tgcctgtgat 36000 gtactttgcattccaattag tgccaaagaa tgcacactgg tgcatggtga ccccaaaaaa 36060 ctggtgtggattagcaaaca tcgcagcctc ctccaacccc tgccccagat gaacaacatg 36120 aaaaagaaaagttgactgct tctaaattat atttaaaagt aaacatcagt gctgggttcg 36180 tggaggtcagcctaaagtgc tggcctgtga atgtgccttt aggggtcttt tgagatgaga 36240 acatgaattcatttgagggt gatgaagcta aaaaaaaaaa aggtgttggc tttctgtgct 36300 gacacctcatccgcggagca ggccagcttg gaagtgtccc catgtcagcc tgcagacttc 36360 tctaccatgctgaatgttct cgtttgctca ctttgggttt agaggaatct gcttttctaa 36420 atttatttatcagctcattc cattaccccc tcctctcctg gtggactgtt cttgaccctc 36480 agcatgtagaagttattctg aagtgctgct aacaggattt gatgctggcc ctggggcttg 36540 gtgacagatgtggctctggt cctgcccgtc tgtagctctg tgatcttgaa cagtttgcat 36600 aacctctgaaagcatctttt aaatctaagt aaaataagaa tatgtgtgcc atgcatggtg 36660 ttgggggaagtctcttaaaa atgaaaattt cagcctgtga atacggtagt gtaacaaaaa 36720 tttgtcttaaaatttcattt tttaaattca aaatgcactt ccttattcaa gcattttctg 36780 agccccattgtctatgagaa ttgtgctcca tcccgggatt gaggaaggga ggcggtcctg 36840 gccctggggcagctcgcggg ctggggtccc ttctgaagca gcctgtggct ggtgtggaat 36900 ccggaatccacagctcgggc ctgagcctgg gcttgtctcc tcctcgctgc tttgaacagg 36960 aaaaggcagttcagcccagc aggatgcctc ttagaactgg gaatggcttg gaagaaaatg 37020 cttatgaagccccagcatat aaaatgtgca aggatgtttc cctgctggaa gcatttacct 37080 ggtatcccttgcatttttac tttcctccga aggccctcac cttgtaatcc acctccccac 37140 agttccatcccctctagaca gcagcctgcc tcacagtggt gtcccaagag cactgggcgt 37200 caagaggtaaaatgcctggg cattttctct gaagcacctg ttaccactgt cccttcaggc 37260 tgtgattcctggccaaaatg cacccattct ttttgacaca cttgggaggg tctgggattg 37320 tgagacttgcagaagggtca gatgtcttga gtcactgaga gggctgaaga aggaatatgt 37380 agtctgcttcttgacatttg ttactggccc gggcctgccc catcagagcc tttgagggca 37440 ttaccaaggtgccccccaga ttagtgtacc tcctcaacag ataggcattg gccaggctgt 37500 cctacttaggccaggaacca ccttgttgtc tgtttaaatt ctatcacatt ttagtgacac 37560 acatatgtagacatgttata tggtacatat gtgtgttatg tgtgcaggta tttatggctc 37620 tgcctagattgaggcatttg ccactttctg agcacttctt acaggccatg cacttatata 37680 tgcacacatgtagagaaaga gcaaatgaac aagtttacaa aaaatcctga aagccaggca 37740 agtgagaattccatcatttg gaatgaaagt aaacttttct gatgcttcag ggccaaccaa 37800 gtgtgcgatgggcgtcagag gtcaggtctc catgtgtcac ctggcagagg gatggtgtga 37860 gcagctcactggaatttgat ttttccagag gagcatgaga ttgggttact gtgaagtccc 37920 agtgtcgctgaggttgtgga gggtttgtgt tttgtgtttg tttttaattc cttctccgca 37980 atgcccaagtgagcactcca cttgagtctc aagaacccca gagtagagct cgccctgggg 38040 tgctggccagcacgtaattc tcaggaacaa acaagtacat tcgaataaag ctgaggatct 38100 cagtctgcagtgcctggtcc ctcggaggca ttctagtatc cctcttagtt ttgttttatt 38160 ggggcagagcctgttgctga aaacagaacc tgtgggtcac cctgactatc cttcccaggc 38220 cttcgagaactgccttgcat gtagtagaga gactcacagt ttgcaggagt ggcaccgtct 38280 ttgaggcagctttcacataa agcatctgaa gttcctttgc taagtcatgt gcccagtcct 38340 caaaggtacctgcagatcac ctgatcgctg ggagtagggc tggtccaggg ttcctcttat 38400 tgtgtgcacacataaggttc gcccgagtcc ggtgggacag agatgtggga ggtgatggga 38460 tgcagcccctgtaggacccg tgaggctggt ggacaggcag tgcgggctgg tgggagggtg 38520 ttaagggagaattccatcca gctgatcagg tcgtgtctca ggaaggacgt gccccctatg 38580 gaggctgcgagacctttaaa cttgctcatc ccttcccgtg tgtcttctct gggcgcccca 38640 gctcctcccttctccttggt ttaatgcccc ttttctcacc tccctctggc aaccaagtct 38700 gcttgtgtcttcctccgaaa gggaaactcc tgttgcccct tctctccctc tgttcctcct 38760 ctccctccttgcttcacctt ccctccgtcc ttttgtttct gctgcctcgc tctcacctgc 38820 atttgaaggatgtgacttcc cagtgggacc tgggttggga tttggtgtca ccctccctcc 38880 ctcagcctcaggacatttcc agagcagagt cttcatcaca ctcctggcat ctaacacaga 38940 gaaggatctggataaaagtt tgttttgttt tctgatgggt aagtggaagg agagagacag 39000 tctctcagctccagggagct gagtgcatca tgcctgcaaa gggcctgagc ccgcagagtg 39060 ggagctgcgtgagaccagca cctgtgtttc tgagaggaga cctggcattg ccggctggtg 39120 gggtgggctgtcctcttcca ggtgtctgat ttgcacccct gcttgatttt gctaaggtgc 39180 agtgggtggtcctggctgta gcagagtcaa catgtggaca gtccaggtcc tgtcgtcccc 39240 ggggatctctgggtgtttgg gtggggggag cagactgtcc gggaagaatt tttaaatgtt 39300 ttttcctcagtggacttgct gccacctgga gcccaccttt gaaatcactt tagtccctgg 39360 cactgcagctgaggagaaag aagcaagaga ggaaagataa acagatctcc agaagatgct 39420 taaatctgtagattcacact caggattgtc ccagtcatgg ctgtgacacg ggcacagaag 39480 cagcatgccctgcctcctcc tccctgctgc ccatccctgt gggtttgctc cgagtcactg 39540 agcccacagttccacggagg gaatgggacc cacccttggg agtgccatcc tggtcacagt 39600 cacccttcctgggcctggct gccccatgct ggtgccgccc cgtgccgcct tcctctctgc 39660 ttggggagtgacaggcacgg gaaaggccag tccatccgct tccgctgagc ctgctctgtc 39720 cgtggtcaggcatttccgac agcatcattt gcaggagctg ctgctctgca acacataaac 39780 aaccagggaggaaatgccta ctcagacctt tgggaggaag gtcccaggga tcttcctggc 39840 ccatgtgcggagcggtggcc ctgagggtcc caggcaagcc gtgtccctct tggggtaggg 39900 cattgacctgcccactcttc acaggttgat gagtgtgcca gggtcgtgca ggggctggac 39960 cggccaatggctcggctcag gctgggcagc aagctgtgct ggaagcacag gcgtgcacag 40020 gggctgtaagaaaggaggca gcccaggagg aacggggacc tgctcttcca gggagagctt 40080 tctaggggaaggggtgacac accagcattt gaagagggag tgggattttg ccagacacaa 40140 gagacaagagaagccaggag tgagtgtctg ttctgtgctt ccccttgtga aaaccctgag 40200 taatgtaccgtgtctctaag gcccagacac atgacaggcc cctatgttct gccatgtgga 40260 ctgggttatagtcagatttc gggtcacggg aacagagcac tttgacttga ctgagggaga 40320 tggtgttccaaatgctgaaa aaaatgtggg gtcacttgaa aagggctttg tagaatgtat 40380 aggagtttttcagcagttgg cttaggagtt tggcctttta gatgagagaa gcatatgagc 40440 aaagaacatggaagaaaggg gacacagtgc gtagaggctg ccaacagcct acagtaggtg 40500 ccagccaggcagaacaggct gagggatcca gaatcaggct ctaggacagg gtttgggcag 40560 gatgttatagcccattgggg cccggaagaa aacctgcagg gtttgagcag tgcagtgaca 40620 gggtgggatttgcattggaa ggtcccctgg gctgccttga ggagtgaaag gcttccagcc 40680 tggtggccaggccagatgag aggcagatgg ccccatccag aggaaaggga catgggtgta 40740 ggctcctgccggggtggggg gcagaggact gactagggac atccttgttg ttagagactg 40800 ttcaggcttccacttcctct ctggctagtt gctggaagcc tctctctgct gtgagtgtgg 40860 ggtcccaggggaatgggggt ttgattttgg aggggcaggt ttgggggcat ggtgggcagg 40920 tcctgccgcttgtgccttgg gtgatggctg gcctgaggct gaaactaagc ctggttgggt 40980 ttcagcagggaggttgcaag aaattaaagt tgggggagga aaaagtgaaa agaaatgaaa 41040 aggaagcaaaccgcagcagt gagcaaggcg gggccagggc cgtctggagg cactgactgg 41100 gcaggccgcccgcgacagcc agcagcagct caacaatccc gggattcttt cccgggcctg 41160 agctctctcggccgccctct gaatgggcct ctttggaggt ggccgccccg ccccctgcac 41220 gactccctcgggacaggccg gaacacagag ccctgcctgg gtgggtgggg gacgccaccg 41280 ctgggacagggactcgggac tctgctcacc acgtagggct gctggggaca cagcccaagg 41340 agttccctctgtctggccca catcctccca ggcccttcca tggactgacc agggtcctgg 41400 gtgccaggtaagctgtacat tgggactcat tgcttcttcc tgctcagtcc ctacacgcct 41460 ggtgtcttctgtgctcctgg cgcctctggc ccggatccag ttgcccttgc tgaaacttga 41520 tctcctgggctccagtggag gggggtgggg aggggtatga ggtgggatgc agaggtcact 41580 gggccaggaatcctgggcag gacttgggag gagggctgat tgttccaaga gtccccttgt 41640 cacttgatgacagttctcaa gtcgccaagg ctttgggaca aaacgcccca gcccttgagc 41700 ctggagggctagtccttcca gaagaaagtc atgctttgtc tttgctgaga ttcactcagg 41760 gctaacaaaagcctggttta cagatgtgca gcttttctgg aacaggactg cttcctagca 41820 cagtgcattcctttactgaa actgataaag cgagcctgcc ccattccagg gctgtcaccg 41880 cccccaccccagttccaggg cccttgaggg atcccgggca gcgggcagca ggaccttcag 41940 aggaccagaaggctgcactt ctgagcaccc agcacagttg tttctactaa atgccgaaag 42000 caagcgttaggatcaggcgc tgggttctaa aatgagattt actgttgtct cttctgtggt 42060 tgaagcaagcaaagagagta tttctaaacg agttcatgtc attgaaaaaa acaaaaacaa 42120 aaacccatgcattcactgaa agaacctgct cagattatgt taacagagct tctggaccta 42180 ggggctttgtttcttcactg tcccttccct cccaaatgtc tgaactgggg ctggggctct 42240 gtgggaccctgggagagtca gccaggcact gggggtagca cgggcagggc tggcgcctgg 42300 gcccagggtgggcctcctgg gcatctgctc cctcctctgc gtggctggcc tgtgattctg 42360 ctggccctgcagccgacaca gcgtgtgtgt gtgtattcca gctggaagga tggggaagat 42420 taagagggagcgaggggaaa acccagagct ctttattgtg aaacgagcac tgcagccgct 42480 ctgagacaagcccaagtggc tcaaggtcca gcttctcctt cccaggctga aaatcttgcc 42540 cagtatcccttccaggattg atggattgct ctccagctga tggaagatgc tatcttttca 42600 aaagtttcctgtccttttaa tgtattttat atacattagt aatttttcat gaattccata 42660 tgcgtgtcagtgtattagag attgtctggt taggacggac atagttttct gactttggag 42720 gtgagattaaaagaattttg gtccacctat ttcgagagag gggtgatttt ctcccttggc 42780 caacctgtagccatggtggc ccgtggggag cctggtgctg tcttctgctg gggcttcctg 42840 cactctaagggggaacctca gtgacagggc tctaagttgc tcccagagta cctgtgcctt 42900 cctaatccccagggcaaacc tgtgcagaag gttccttgtc ctcctgtagg ggcagagtaa 42960 ttgtattacattctgcgttc tgactcagac cctgctggtg ggaggcaggg atgtcttgtg 43020 gaaagaacatagttttctga gtccaacaga tgagttggct tcctttgaat aagcttctta 43080 acctcacttttcagaatctg tttcctcatc cataagatga tttgcatgtg gaaatgtttg 43140 tatgaggattaaatcagata acacctatgt gtgggttagc agagtgcctg gtgtatagta 43200 ggcactcagagaaagggagc tgtaattctg atttccattt tactttcttt atatgaaaag 43260 ttgatatgtgtcatataaac aggattttac aacagtccca agagctgtgt gatttaaaag 43320 ctgttgacattcagattggc acctgtactg atggcaacat ttctccaaaa gcacagtgaa 43380 agctcccgtggttggagctg ggatctgata atccgcatgg ttctaaaaag gcagtttgca 43440 gccaggaccatgcatcaaca tagttctaac tgctgcgcac tttgaccttc tctcctctac 43500 ccttctgtttccacttccct gtctttaaaa aaaataacat ccaaggcaat caagctgcct 43560 agttgatgtaggatcaggac aggaagcctg gtgggtgtga tgcatggaag gtggtcttac 43620 tgctgtcagacacaggtctg ggatggctcg aagtcctgtg gtctgatgag aataccatgc 43680 ccattttgtttacattaggc tcacctgaga caggtggcct ctccctccta tcaggaggga 43740 ctgtaaacagaatgagttta cagtaagagt caagggacca gaaggtcccc gtcacacagc 43800 acactctgccttcttgctcc gtcccattgc tcactgtgat tttaccagga gataacctaa 43860 gaacgcaggactaagaaatc actggaatgt caacttctcg ttccaccacg agatagcaga 43920 gtggcctgtggccttccttg agcagtgttc tctgtggaca tatgattctt gattagcctc 43980 aggtagaatggatagctggg tggagaaggc aggtgggtac cgggtcaccc aagtaaatta 44040 aagttaaacagggttttaca aacgtggagt ggaagataga ggaatgaggg ctcagctggc 44100 aggaggacgagggtcaccag tgggatgcct ggctttcttg agtgaccaag tacagataga 44160 aaagcttctagaatgctatg gaactcattt actgctctgt ggaggtaaga aggaaatggg 44220 accaaagataaggcttgctt ctggaagatg tgggtcttgg gaacccctct ttgtgcatct 44280 gttgatgggagtaatggata cacaaaacca cacggtccat catgcttcca agctgactcc 44340 tccaagtctggtttcccttg atagttttcc cagtggtccc atcacttctg ctgcctacta 44400 gatttatcatcacgagaaaa gtcccagttt cctttgtagc tcatgctgca cttgactgga 44460 ggaaatcaagtcaaggatat cttggtgaca attgaaggga tacaggaatt atacagttat 44520 gcgcagactatttaaaaaga cactattttt ttcttaacag ggacattttt caaagttttt 44580 caaagattttcaaagttttt attaagagac ttttaaaaaa tatttattca tttatttaga 44640 tggagtctcactttgttgcc caagccagag tgcagtggca tgatcttggc tcactgcaac 44700 cttcgcctcccaggttcaag tgattctctg gcctcagcct cctgagtagc tgggactaca 44760 gatgtgcaccaccacacacg gctaattttt gtctttttag tagagatggg gtttcaccat 44820 gttggccaggctggtctcaa actcctgacc tcaagcggtc tgcctgcctc cgcctcctaa 44880 agtgctgggattacaggcgt gagccacctt gcctagccct gttaagagac atttaaaaag 44940 ataaaaataaatggagagaa agccatgttt gtggatagga agacaatgtc acaaacatgt 45000 tggaccctcccaatttcatc tgtgtactta atttaattcc agtcagagtg ccagcagatt 45060 attttgtggaacttgataag ctgattcaaa atttatataa taaagtaaaa attctcagta 45120 gccagtatacaactactaga aatgaggact taccacaaag atagaaaatt cggtaaacat 45180 gagctggtacaaggacagat aaattgacca acaaacagat gaaagtgcaa aacagaccag 45240 cacatgcttggaactttggt gccagacaaa acaggcagcc aggtcaggag gggaaagagg 45300 agctgcctcataaatagagc tgaggtttgc caggtagaaa aggttaatgg atccctttta 45360 taacacatgccaacatcagt tccagatgga ccaaggatgt caatgtcaaa taaattctta 45420 gtagaaaatccgggtgcata cgtttttccg gattttttcc ttcctgtctt ctgctcttct 45480 gtttctcttttcctgcctta ctgtggattt atttatcatt taggggaaac tctgtcttta 45540 tttattgatagtatttttga gtatatccct ccctgtagtt tttttagtcc tcgctcttgg 45600 tatatacataacttgtcatt atttatggct acttgtatta ttttatcact tggagcgtag 45660 catagaaatcttacttcaat ttaagtttct tcaccctccc catttttaaa gtacaattgt 45720 tggaagtatcttatccgttt atggtgagta ccacatgtga tgatgtctgc tagttgctcc 45780 aactataaaaagtgacttaa gaaactcatg agaagaatgg actgttatat ttacccttac 45840 tttgactcatttgaacattc ttctttcctt ttggaagttc caaatcttat gattttcatt 45900 ctgtttagaaaatttccttt ggccattccc tactgggaca tttgcaaaca acaaaatctc 45960 ttcattttccttcatctctg gatttcttta tttttccttc attcctgaat ggtatttctg 46020 caggatatagaattcacagt tgacagttct tttctttcag taccggaagc atgtgccact 46080 tctttctggcctctttggtt tgggtttctt ttgaagattc gaagataggt ggattctcag 46140 aaaagctgtcattttatatg acactaagat agggaaaaca gtgccaggta aaacaggatg 46200 cactgtgggtgaaaaggtat gcacagattt cagaaatgtg gctccgtgag aaaaagcaac 46260 tggggactgggcacatgagg cctcgaggag cggtacctcc agccctagac tggctgcatg 46320 gtcggcccagggtgtgcaga gctgcaggtg ggctggagac ggggtgccta ccatgcaatg 46380 aatcctcagcaggggacccc tccctgctgg cctcctatgt aggaagccct ggtgatgtga 46440 cagtgaatgagacacagcct cacctctgac acggttccag cagtgtgcta ctggccatgt 46500 gccagatagagcagggtccg tggccaccga gggcctccgt aagagggaat cagggagcgg 46560 aggcaggctgctcttgaggg ctttagaggg cttcatgccc cgggtagggc ttctgtgatt 46620 ctgcggggtctccagtcctt ccagcttgag cactgcactc gactcatggt aaatattatc 46680 agcaacttagaatatttagg cactcccctc agttttatgt tcgtcatata caggctttca 46740 caaaccattttcttttttct ttttctaatc ataacattcc attcctaggg tatgtttgtt 46800 aattttcatttcactgggtt gtttttttga ttgtgtgtgt gtgtgtgttt gtttgtttgt 46860 ttgtttgatacggagtctca ctctgtcacc caggctggag tgcagtggcg cgatcttggc 46920 tcactgcaacctccgcctcc cgggttcaag cgattctcct gcttcatcct cctgagtagc 46980 tgggattacaggcatgaact accacgcccg gctaattttt gtatttttag tagagacggg 47040 gtttcaccatgttggtcagg ctggtcttga attcctgacc tcgtgatccg cctgcgtcag 47100 tctcccaaagtgctgggatt acaggcgtga gccactgcac ctggcctttg cttttttttt 47160 ttttttttttttttgcccgt tctacttcac tctcatgtgc tttcccattg gtttgtggtt 47220 ttgctgttgcatcagcttcc ttctataggt caaggctatg taatgtaccc agaccctggt 47280 cctggggctgagggaccagt tctgagggat gcttgatgct tttacagttc tttagccttt 47340 aatctttttgtagaatgcca atgagactcc tccttccagg acagctggga gggtcgggaa 47400 gtcctcagtgaatgttcatc atcaggaaaa ccttggtgga tgatgggcag gagggaagct 47460 tcagcagtgggtccccctcc ctgaactgct ggttagagag aggcttgagt gctgtagaca 47520 aggaggtcttttatattgtg ttgtgcattc taagtggatg gtctttgggg gaagaaaaca 47580 atgtagagtaaaatcaggac aatttggtct atttgagcga aggtctttct gaactcccaa 47640 gtgtttgttccctccttccc ctcagagcac aatggttatt tcttaaattt atttcctatg 47700 agacaggagtaattatgggc agagggacag atgagagctt caagcattgt ccagttctgt 47760 cccgggacagccctcttgga gagagaacca cctgtggcag agactgtgga ttgctgtaaa 47820 ttatcttattaattggaaca tcacttgtta tattggagca attatagcat gttcaaagct 47880 ttgctggaaggccctccaaa tgccgacaca ccctgctggc atttcagtag tgctggtggc 47940 ctctgtattcgtgagttttg gactcagtgg aatggagctg ggctccatag caccttcggg 48000 gtccagaagccagtggcatg gtgcatgacc gtggagacgc tgcccatgaa gaacatagag 48060 ccagcctgaaggagaatggt accaccgaaa accagtgggt tgagaatgag acccttgcat 48120 cagcaaagtgagtagcctac ccattaggca tggtatgaat cggaaacctt agtgaaacaa 48180 aaacacaaagagaggagaat cgggaccaag aaggaaggcg ggtgaagccg catcatctcc 48240 ctcctcgggctggctcaggg ccccatgaga tgcaggtttt ctggcacaat tggctcctcc 48300 tggagtcctgcctcaggctg gtcagaattc ccatgctcca agagtcagaa gggacacatt 48360 agaaagtctgtgtggtctcc tcaaaagcca catgcctcat gtcagttttc atttcttcct 48420 cgtcgcgatggatctttcga gtgcttgcca ggggccaggc cctgctgtaa acacctttca 48480 tgccctaacttggcagattc cctgagaact ctgaagtagg gcctatgtca ctctcctgcc 48540 atttatagaggggctgcatc tccccaaacc acacacaatt cgtgttaggc tgaattgtga 48600 ctccctccaccccctgcaca ttcctatgtt gaatgatgcc ttaacccgaa ccccaggatg 48660 tgagtgtttggagaaagggc ctttacagtg gtgactggtg aaagtgggtc ccttagggtg 48720 agccctcatccaacatgact tgtgtccctg tagaaggggg cgatcaggac acagacacac 48780 agagggaagaccatgtgagg acacagggag aagatggcca tctgtgcacc gcagagagaa 48840 gcctcgggagaaaccagccc tgccaatgcc ttgatcttgg acttccagct gctagactgg 48900 gaggggatacatttctgttg gttcagtcac tgtgtggtat tttttatagc agcccaagct 48960 gactaatactcccaactcgt ggaataccag gattcctctt aactcacaga gctgtggaca 49020 gagtgaaaaggaaaaggaat gaggttcgtg ctaatgagct agaggtggga aacccaagaa 49080 tcatgtttgagtcctgctcc agctgggtgg tgcgtgtgtt tccctccagg tgcaggtgag 49140 gggaagtgctgaagcctcgg cctcctcaca ggtatagatg gacgagagtg cacaccttta 49200 agagttgttctgagggtcaa gtaagcccac tgagcaccgc gcagatagtc actattagta 49260 aaatagtgataatttataaa gataacttat gttttctgaa caaagataaa atgcattctg 49320 agtctctgtttatatttact gcaatgagag tacatctttt tgttaactga gtgaagggtt 49380 taagtgaagctgaggtttcg aattcttacc taacacagga ttcccagtgt tgcattcctc 49440 attgctcacaggacgcttta gaaatactgg gtgaagatgg caacgcctgg ttagggacct 49500 ggttagagtctgaccagtga tattctctca aaagcagcag tcttccagtt tctgaaacta 49560 catgagaaacccccattagc tgcctgtggt ccctggcaca tgggctggtg ggcataaggg 49620 atgcagatgccccgagtgcc acaggggttc ttagacaggc aggtccttta agggtgtctt 49680 tgattctattcaggtccatt gaaatctgcc acatagaata aggttttcaa gtcatggtaa 49740 gagacttctgagattacagg ggaggtttct ccccctgggc cttatgtgat ttagacttcc 49800 atggtgcagtcattgataca gtttggatat ttgtcccctc catgggggca gatacttcat 49860 gaagggcttggtgctgtcct catggtaacg agtgagttct cactgttagt tcacacgaga 49920 actgattgtcaaaaagagcc tggcacctcc cctcctctct ctctctctct ctgtctctct 49980 ctctatcactctctcactct ctctctctcg ccttcctctc accatgtgat gccagcttcc 50040 tttcacaagtccagtgggag tggaggtaga caggaatgca ctggcatggg gtggttaggc 50100 cagggtgagggtgtgcagag tgcacatcaa tggaagcttc ctgaggccct caccaggtgc 50160 agatgctttttctacagcct tcagaaccgt gagccaaata aacctctttt taaaataaat 50220 tgcccagcctcggttgttcc tatatagcgg tgcacatgga ctcagacagt ggttgtagga 50280 caaaatgatgatcctggacc aggactcctc gggacttcag ctgttgagcc accaggctaa 50340 agtgacctttccttagaagg gctgtggcaa agttcagcac atgtcatctg tgtcccatgt 50400 gccgtttctaaaggaaagcc agattctctg ggaattctgc ctgaaaacag aaaacatcaa 50460 gacttcctgtcctgtggaca agtccagtta tgagactgga attattctgc ggactaaaga 50520 cttggaaccaagcatcttag gtacagcctc ttcattggca tagaccctcg ccggtatcat 50580 cttgagctgcctgaagcttt ctctgaccgc tgtccactcc tgtgtggacg tggtgtatac 50640 attatggtaacctccattct ccatcatgat aatcagccca gggctctcaa agcctcttat 50700 gcaggttctctctctgcttg tgaagggcaa tacttcctct gtttgtgggt ttttgttttt 50760 gttttgttttggctcagcat aaggaaagct tccctgagga gggtcagatg gctcttttct 50820 tttctggtgctgctttgcat accctcaccc tggcctgact gccctgtgct ggtgcattcc 50880 tgtctgtttgcactcccact ggactggaag cagctttgag gcaaggacat tgtgtcactg 50940 agtctgtttcctgttgcaag taaccagtcc caactcaaac tggctaaagg aacaagagag 51000 gagaaaatgtaactgttcgc aaagtcaaag ggtgaaagct atcgagtgtc cagggacagc 51060 tgaatggataaacaaaatgt gatctatgca tgcagtggaa tattactcag ccttaaaaag 51120 gaaggacgttctgacacgca ctacaacatg gaagaacttc agggacattg tgttcagtga 51180 agtaagtcagtcacaagaag acaaattctg tatgattcca cttacatgag gtccctagag 51240 tcattgaattcatacataca gaaagtggga tggtggggac ttgggtggag gataggaaat 51300 tatttcaatggggaaagagt tatagttttg taagattata aagttctgga gatagttggt 51360 gtacgaaaaagtgaatgtac ttgatgctac agcactgtac acttaaaaat ggctaagatg 51420 gtaaattttgttatgtatgt cttatcacaa tttgagaaaa ataacgaaac tcactgagca 51480 gctgtgggtggctgtggcct cagatttggc ttcatgagga ctccttggcc actcagcggg 51540 tgggtctctctctctgtctc tctcacacat ttatttccag tgactcccta gaccctgatc 51600 ttctcagctgtgtgttggct ctgtctgcag gcttgatgtg gtctctaggc agcctcagta 51660 gctccacttcagtgtcatct ttctttaaag tctagaagga aggaagtcat ttgttttctt 51720 gagaactcaaaccaaagcac atttttttac aactctcatc ccttgtaggt tgctaaggga 51780 ctatgtgagtccctggagga actggcctca cttagtgcat cccaagtgta aaaactgctt 51840 gggaaacactcactatgaag aaaaacgctg ttttcactgt ttcaacatta gaattctcta 51900 cacaacaccaactaaactgg taaatgtgtt ttctttccag tctgatcaaa accttgaata 51960 taaatgggacatcttcaaag aataagagaa aatagggctc caactgtaat gaactattgc 52020 atctacattttctacttcct tttgtgttat tgcttaaaag gaagactgtg gcatggtctc 52080 ttttgaaggggataattcca attgtgatga gaatgtcata aaagggtcac ccggcttttg 52140 gtggacaggattcccatttc agccctggct ggagcaattc tggcttcact gggggaataa 52200 gaaccttgcttcctttaaaa agacagaaac aatttatcct tttgagagca tttctcatat 52260 ttgctgaaatcagttgacct taagctgtag aacaaatttc atattatttg tattatgttg 52320 acatcaggattttaaaaaga aaagattcaa caccaagaaa gtatttattt atccatttgt 52380 gggtttttcatcatttttgt ttgtttgttt gtatctgtag gactttgatt ctcatcggaa 52440 accatgaaaatgcttttcca gtgttggggt ggtcactaat taattctcat tatatctggc 52500 tgcctgccattttctctttg ctttcaagaa ggttggttaa atggcctgat tgttcataga 52560 caaataacaaaagaattata ggataatgac tttttttttt tttttttttt gagatggagt 52620 ctcgctctgttgcccaggct ggagtgcagt ggcacgatct tggctcactg caacctccac 52680 ctcccgggttcaagtgattt ctggctaact tttgtatttt gaatagagac ggtgtttcac 52740 aatgttggccaggctggtct caaactcctg atctcaagtg atctgcctgc cttggcctcc 52800 caaagtgaagataaggactt taaaaaaaaa aaacaaaaac aaacaaacaa aaaaataaaa 52860 ctaatcactactgaggaaac tccaatacaa agtcttgctt ttctaaatta gaatttaaaa 52920 attcctagctaaataaaccc tagaaatcat ctaatctgat cccaattgaa gtgagtctgt 52980 aaatgaagatcctgaaggcc ccaggggttg acaatgtgcc caacacccca tggtttatgg 53040 cagaagagctcaggctagaa cttggagatc tgagctgttt ctctgcagca gccccaattc 53100 cccgcctagaaatgtaatgg aagaaggata cgagagattt gatgttccat ttttttagaa 53160 ctctctcaaataatggaaaa agatacttcc tatttaactg cataataatc ccgggatgtg 53220 actaggagggaggaaagaat ggagagagag agagagagag agagagagag agagagagag 53280 agagagagagagaaatagtt agttaggtgg atagtctcac ttacttctga aagtttattg 53340 tgaatatgactgtgtattta tgggccaaag attgagactg tatttttagg tagttaacaa 53400 aactcaagtttcttcacttg gataggagac tataatattg acctcagcta aatttatttt 53460 atttttcattaaaggtgata taaaacattc aaaggcgatt tgtagttact aattgatgta 53520 tgagtgaataatttttatgc cttatcagga acacgggaaa ttgggaacac aggaaaatgc 53580 tgattttgtttttaatgtca catgtcctga ttttttgcta agtaagttat ggttcctgag 53640 gatgaactttgatgtttgga tccctcatga atgtttaata tgtctgacaa ctccttacac 53700 atttaggttgcaggttttgg ccaggctcta aggcttccga agagcgagcc agtggccctg 53760 cctgagccccgtgggtttct cctcttcctg ttaacttata tgtcatgtaa atagtggccg 53820 ttggagaatgagtggtatcc agcttcccac agaagcctgg atgagagcat aaaaatagat 53880 cgtttggaaaagggcccgtg accctctgtc acccttgttc tgggtaaagg aaatgaagga 53940 ggtggaagccgagaggagag gagcatagac attcctgaag tcccagaacg acgggcacgc 54000 gcctcccaagttctagcccg gtcccggggg gctgccccgc agcagcagcc ttcccggcca 54060 cctcacccccgcagggcacc agggacacca gctgcttagg cggaatacca gaaagtgaga 54120 tgagtgttgtttccacacac taacggtatt atgcctcctg ctctgggtct gagcagcaaa 54180 gggaaaatgtgagaatattg tgggaattaa gggcagaagg aggccgagcc agcaggatgc 54240 tggaactaaaaggagcgtct gttcagggcc caagatcggc tgtggctggc agggctgtgc 54300 tggcgtgcacacggtctaaa cagcatccct gtgtggcctc tggagcctcc cacgttgtgg 54360 aaacacatcttcatagggga ggggtgcatg gcctcaggaa gacggctttt gccttagtag 54420 aagttggcctgcaccggaaa accctccatg tagttgcttt gagggaccat tatgctcact 54480 gtggtgcccttatgtctcca cctctgaagc agagagacag gctgccacag gcgcagggct 54540 ctggagagagcagcgggctt aatgcctccg cagatgcccc tgtccaagac ggtgcaaatc 54600 ccatctgctcctcaccccgg tcatccccac cccgggccat ctcctgggct cttctaggta 54660 cagagctgaggatacaggca cctgatcgcc ccacaacaag gagctcagcc cacgtcctgt 54720 cctcatttttaacattgcta aaaatgttca tggtagttta accgtgaaca tcaaatgtac 54780 aagttgagcaacaacaacaa ttctgcctgt tcttatggtg agccacattt ctagaaatga 54840 agtgaactcatgcaaaaggg tttgcagtag gtagatcatc ctcacttgac tggtgcattt 54900 gactatacgatttttggtgc acctccccca gccctgccgg ctcctgttct gtgcttgcta 54960 gatgctcagcagggacagtg cagaggagca cttagaaagg gacccaggct cccgctgcct 55020 cgctctcctcttccgttaag catgggcaca tctcgaggag ctgtgcagac ctaagggaag 55080 tggagatggtaggtgccaag cacagcgcct gcagggtgta ggttctgtgt aaagtgtagc 55140 tgctgtcagcatcctccttg ctgtctgcag tcccagtcac tggaagtgac tgcactgacc 55200 tgaagatgtgacactcaccc aacttatctg ggaatgccaa ggcccatcta ataagtggat 55260 taattgctgtggcattgagc tgtgtggcac tctgttagcc gacttgcatc tctgagcaca 55320 ggcaaagaccaaggaccttc aggagaaatg gcccaggaag cccaagtctc cctgtgctga 55380 agtccttgggataaacatgg aatctgatgc tttgagcagc cccttctcca aataaagggt 55440 taagtcctgtcacccggtca ggaactggtc tgacagaggg aactacacag ccagagtgcc 55500 ccagagttggtgctaattga atgataaaca aagccacttt catgggaatt gctgggcttg 55560 ctgttttctgggacaagcac tgtgataaca ggaacaaagt ggattttatt ttccttcagc 55620 ctcttctcatttggtgaaat tcccaggcgc catgggagtg cattgagggc ttctgagtca 55680 tggcatggggtcttctagag ccttgaggca cggggactct gtaaacaggg atgtgaagca 55740 ccagggattgcccccgtggc tttgtgggaa ccactaagag caatgtcagt catagaaaca 55800 ggtactgcatgctgtgctgg gtatgtgctc ctctcttata tgactctttg aaagctcaac 55860 caaccaaccataaggtgggc atattaaact tgattaataa actttttttt attccaaagt 55920 tctgtgctgcttcttggcat gagaccctag gaactgatca accaactgtc cacctactcc 55980 tctgaaccagacccctgagc agtatttcca cgtcatgctc tgaacctctc cctctaacct 56040 ctaagcctttgctagttcat tcattaattc acaatgtgaa tgacatggag ctcctacagg 56100 tgccagcccagtgctgggga cacagcagtg aatggcagga gtggcccctg cccaataagc 56160 aaagaacccatgaggtggtt ttgtcagaat gcatgccacc catccttcca ggatggcatt 56220 catcaaatgccacttgctcc aggaagcctt cagatgaagc tccctccaga tgaagcaagt 56280 ctccctgtgggtcctcctgc agctcaagag ctgcacctca catgttcctc catgcagtag 56340 taatttgcacatggtctcat ctggctgaca caagcaggac acgcaggcac tccctgtttc 56400 gcttttgtttctctcttccg ttctccggct ggtgtaggcc atgttagtgg aaaactggtg 56460 cccagcaaagacattttatt ttttattttt attttttaat tttattatta ttatacttta 56520 agttttagggtacatgtgca caacgtgcag gttacacatg tgtacatgtg ccatgttggt 56580 gtgctgcacccattaacttg tcatttagca ttaggtatat ctcttaatgc tatccctccc 56640 ccctccccctgacattttat ttttttattt ttatttttat tttttgagac agagttttgc 56700 tctctcgcccaggctggagt gcagtggcat gatctcagct cattgcaacc tctgcctccc 56760 aggttcaagtgattctcttg ccttagcctc ccgagtagct gggattacag gcacacacca 56820 ccacacccagctaatttttg tatttttaga agagacgggg tttcaccatg ttggctagga 56880 tggtcttgaacttctaacct caggtgatcc gcctgcctcg cctcccaaag tgctgggatt 56940 acaggcatgacccgccgcac ccagccccag caaagacatt tttaatggaa aagaaatacg 57000 gacaaacacaaatggagata agtcatctta gtactgggaa tagaagtgag ggtgcagagg 57060 gatggttttaaactccatgc atctttgttt ttctatccca gctttaatag aaagaaataa 57120 taaattcagcaaacacttat tttaggacat ttcctgtggg ccatactggg aggaggggcg 57180 gtcagtcacacaactgcatc tgctctatgg atttacaggt cagtcatagc agaaaacgtg 57240 cacatgtattcatgacacaa actgagcatt tgccaggcct ggccttgaga tggtcatggt 57300 atttacagttgggtgcagca gacacaccat tcccaagggt cttaacagta tgttcataca 57360 aacagaacattagtactggg cttttctata atggaaacat gattaaatgt tgaaaacatt 57420 agtgcggaaattggtgtctc gtcctttccc ccaaaatgga caaggcactg tgtttcagac 57480 tttgtatttgatttttcaga agttttacta ttaagtgctt atgtgtaatt ttctttttat 57540 taatccttcttaggtttttt gtttgtttgt tttgtttttt aacttgtggc ttgatttctt 57600 gagtcttttaggaaattctc agtatgtata ttttccaata ttgcctctgc ctcattcaat 57660 ttctccacaacttcttggac tccaattata ggtatgttag aatttttaag aatcatttct 57720 catgtcttactgtcttttgt gtatttctca gtcttgttcc tctttatgct tcagactgca 57780 tattttcttcctatttttct cccagttcac tcacaatttc ttttgctgtg tttagtctgc 57840 tgataaaatcatgaactgaa tgcttaattt catttatttt gttttgagtt ctagagtttc 57900 catttgattattctctcttt ctctctctct ctctatatat atatgtatac acacacacac 57960 acacacacacacacacacac acacacactc tttaagttct agggtacatg tgcacaatgt 58020 gcaggtttgttacatatgta tacatgtgcc atgttggtgt gctgcaccca ttaactcgtc 58080 atttacattaggtatttctc ttaatgctat ccctccccct gcccccagaa tggcaatcat 58140 taaaaagtcaggaaacaaca ggtgctggag aggatgtgga gaaataggaa cacttataca 58200 ctgttggtgggactgtaaac tagttcaacc attgtggaag acagtgtgga gattcctcaa 58260 ggatctagaactagaaatac tattgaccca gtgatcccat tactgggtat atacccaaag 58320 gactataaatcatgctgcta taaagacaca tgcacacgta tgcttattgc gaactattca 58380 caacagcaaagacttggaac caacccaaat ttccatcaat aatagactgg attaagaaaa 58440 tgtggcacatatacaccatg gaatactatg cagccataaa aaagaatgag ttcatgtcct 58500 ttgtagggacatggatgaag ctggaaacca tcattttgag caaactatca caaggacaga 58560 aaaccaaacactgcatgttc tcactcatag gtgggaattg aacaatcaga acacctggac 58620 acagggcagggaacatcatt tgattattct taatggactt cagttctttg gtgaaattct 58680 ttttgcttctgctttcttaa acattctgtt cagttatgtt agtatctgtg gcttttaaca 58740 tcaatttttgaactagctgt gggcgttttt tctgtgtgtg gggtggtggc agtcttggtt 58800 ttcatttgtcatgtttcctg gcattgatga tcattttttt acaattaaat atttgatact 58860 gtatttttagagtatagaca aagtcaggct ctgggtgggg gaggcatgtt ctttagagag 58920 gatataatttttcttccagg ctgcctaatc agatcctgtc atggtcctct tgtttctagt 58980 ttgtagcccttcaagggtct caccttgaaa ccttgtggtg tttgcaaagg cccctcttcc 59040 taggtgcatcttgaactgcc gtttttggtt acttagcatc cactaagaga ctgccacaat 59100 ccctgctgatcttttttaga ttcgtagcag ctggctttgt cttgtttact gggcattggg 59160 ccctgtacctctgccactta ggactcagaa atgccttgag gggaaacatg ctcagagtgt 59220 tgactctgtctctgcagtcc tcatttctct gagaccttgg cacttccaga actggtgcct 59280 tggtagctctgaactccaaa cttggtctcc ccagaacagt gtgattacca ctgctagttt 59340 gtgtatggcccctataccac atgcttccaa acgggtccat gtctgagaag gaaaaatggc 59400 catggatctcctgctccctt ccctggaggt tttggcccag taagccctgg ctgccttggg 59460 cgatctgaatgggttcaaac tattgttgtt agtattttgt gtagctttca taattgtcct 59520 tagtggaagtttaactcctg cactgtaact gaaaattaac atgctctctc tttctagtac 59580 tggctttgagctgatgcagc tgatgtggcc atctttgata gataagggtt tacagattgt 59640 aatgtctccaagattgttga taagcatgtt tttccacctt tcttgacgac ctgcagtgaa 59700 caatctaacattctggcttc ttctacctca ctggataaac atctgtttaa cctctttctc 59760 tttagccataacattgaaca tttgtgtaaa ttattcatcc tcccttttag cctactaatg 59820 aaccctctgtggtgggtagc agtattaatc ctatttatcg gtgaggtggt aaactgagaa 59880 aggtaaaacaaattacttta aattcaaaca caaacctccg gcttgttgag ctatttttat 59940 tattccacagttatgtttta aacatactct catagcatat tttttttttg ctctttttca 60000 ttttaaatactcttaagtct tggggatatt aatttataga gttttcaata atttataatt 60060 attgaataattgataagtta actttaattt cagtgaattg ttcgtcttta aatatgaaag 60120 attagagatgtatgcttcag gtcaggatgg agtaacagag acttgattta ttatcctgac 60180 tgaacaaacaaaaaatggac aaactatatg aaataatggc ttccaagcca atgggcatca 60240 ggcagtgaaagacaacgatc cctgaaaagt aagaaacaaa catggtgaca gtacagttgt 60300 cccagtttaccgcctagaca aagtttccag gtgtaaggca gggagaggaa actcacgcag 60360 aatctgggagactctgagtt gggcatttgg agttgaaact ccaggggacc acaatgactt 60420 gcaggatggagagaaggctg aacagagaga gagctgttgg agatctgcag aggatccgcc 60480 atacgtattcaactgagcgt tgggtaggag gacattaggg acattgaagg ccagggaaag 60540 aaccaaatgaaaagattgga aagagaggct aacccagtgc taactcaaag ctgggaagag 60600 tgcctgttctcatgagcaag agaggaaagt atcaagattc actgggcatt gagtgatata 60660 cagaagggttttgcccagta gtgggggata acttgcacta gatggagcat gactccaggc 60720 cttcctaacaaatgtgaaga gcaagaccca aaaagataaa aatgtttcta agcaacttaa 60780 ctgcatcccagaataaagtt caagaatatt tataagaata caaaaatatc cagcatccaa 60840 aaacataaaatcacagtgtc tggcattcaa tcaaaaatta ccaggcaatc aaagaagcct 60900 gaaaatagcaatatagttaa gagagaaagc aaaacaaaac taatgaggac acaaaactaa 60960 tgttaagattatgaacatta agacaaggac attaagacat ttatttttac tatgtttaat 61020 gtgttcaaaaagttaagtag agtcgtggaa catataaaaa gacccaaacc aaacttcact 61080 ggagagagaaactccagtgt ctgaaatgaa aaataaccta gatggattta acatttggct 61140 gattttggataaaattacaa aagcaattca atgaagaata gatagtctca acaaatggta 61200 gtggtaccattgaatatcca tatttaaaaa actaaacttt gttgatatat atgcctcaca 61260 ccatgtgtaaaaattaactg aaaatggatc ataggcttat gtttatagtt tcttccaaaa 61320 ctattacacttttggaagaa aacagaaaaa catgttttgt gactggatta ggcagatact 61380 attttagatacaacaccaaa cacactattc ctaaaagaac aaattgataa actggactta 61440 aattacaagattttgttctt caaaagattc ttaagtgaat atatgcacag gcaacagact 61500 cagaagaaatatttgcaaat catatatatg ataagggact tgcatctaaa atatataaag 61560 aactcttacaactgagtgtt aagaaagcaa acaacctagt gaaaaaaagg aaaaggattt 61620 gaacaaacattttaccagag aacacataaa aatgcctatt aataagcata caaaaagatg 61680 ttcaacatcattagtaatta ataaagttgc aaattaaaaa cacagtgtta taccactacg 61740 ccagcactagaatggctaaa attcgaaatg ccaaccatac caagtagggg caggattgat 61800 ggtggggatgtgaaatgctg caagtattct gggaagcagc tttgcatttt cttaaaaagt 61860 tacatagacacttaccatat ggtccagcca ttccatccct aggtattcac ccaaaagtgt 61920 ctaaaaatactcattgcagc tttatatgta gcagccccat agtggcaaca acccaagtgt 61980 ccatcaacaggtaaatggac agagaaaatg tgatatgtcc atacaatgga aaagtactca 62040 ccgttgaaaagcaatgagct atgtatacat tctacaatgt ggcggaatct caaaataatt 62100 tcttaagtgaaagtagagga aaaaaagagt ttatatgaat gattttattc atgtagaatt 62160 ttaggaaatgcacactaaag caaagtgaca taaaccagat gagcaggaga aggggctgga 62220 ggaaactcttggaatgatga tgtaactgtt aattatctca atcatgatga tggtttcatg 62280 gacgtttacatatgacaaac tgaccgaatt atttacttta agcatatcca gttttttgta 62340 tattagttatacctcaatat ataaaggtgt ggtttttaaa gtttcatgaa aaattaaaca 62400 tttaaagttcactttttctc taccaaggtc tattagataa cttaggatag ttgttaattt 62460 aggcataactccagacattt tgtgtgctga ttaatatatt ttaaaattct aggccacgcg 62520 tggtggctcacacctgtaat cccagcactt tgggaggcca aggcgggcag atcacgagtt 62580 caggagatcgtagccatgct tgctaacatg atgaaacccc gtctctacta aaaatacaaa 62640 aagaaattagccgggtgtgg tggcaggtgc ctgcagtccc agctactcgg gaggccgagg 62700 caggagaatggcgtgaacgc gggaggtgga gcttgcagtg agtcgagatc acgccactgc 62760 actccagtctgggcgacaga gcgagactcc gtctccaaaa aaaaaaaaat tctctaaggt 62820 ttccttaggaaaagaattcg tgctaaataa aaatagtaat ttttaattca tattgaaatg 62880 acgtgtgaccaaaaaagtgc ttcacttcac atatgtgggg ataccatata atttttagaa 62940 tgttcattctgtcttggtag agatggagct tgtttacctc attggagtga agtgcattcc 63000 atttgtgaattgatagtgca aattctgtag agatcaattt tttttaaaaa accaggatat 63060 tacatcctgtatgtggaggt aatttagttg agtggtcagg gctttccctt gctttttttg 63120 gatataattcctcactggtc tgaataatcc acagaccagt gctcattggc acctggatta 63180 atgaaaaccccctaggtatt tcatcttcag aaatcacata tgctgttgat aattcctttc 63240 agagtattcttagtaaatgt catttttata aagtgtgatt ctggtattca gttttattat 63300 tggttttcatttgaagggta aagaaggtag cctttttttc agtataatta aaataacaca 63360 tatttcaagcatgtgaggtg atagtcctgt gagttgtagg gaaaacagca ttatttggaa 63420 atctctaaatttcaataccc tctccaggaa atgaataata tggagttttt ttctacatta 63480 atattctcttatcaatcctc attagttttg aaacagaaaa caaactgacc agtatttcta 63540 ataatattattttactgacc atggtagatt tgtccatttc ttgaagcctg gtttaaaaaa 63600 agaaaaaatataaaaggtaa gatgttttaa atatccagaa ataatgcagc tgctgggcaa 63660 gaaaccaaacagaaattctg tcagaggaat gtgcctgggc tcaggcatgg gacacaagca 63720 ggtttgcgttctggcccacc tgcgagggtg gcttctgcac cccacggtgt ggcctttgaa 63780 gcagcggggtttgacctgga aagcagcagc atgggtttgc accctcatag tgctcgatgt 63840 tgatggcccagttaccagct ggggggaggg tagggaccaa aggaaggtgg aggacaccta 63900 ggccgcccggctgccgcagg gcctgcactt atggtcttta ttgggagaag tcgcctgctg 63960 gtttgcagaggaaaggccat gccaattcta aagtgaggtg cagcctagat catggagtgg 64020 agagtacattacaggacatc agcctttgtg ggggatgggg ctgggaggac cagcccaggc 64080 ggcccaatgtgcatctcctc acatgaccag ctcgtggtaa tgggctggac actgtggggg 64140 actgaattaggtcgctttat ttttggaaac agtgatgggt gagaataaac ccatgcttgt 64200 gggtgcagggctggccgcct cctccagcct attacgggta gtgatgtcat gttcagtatg 64260 gccatggatgacagaaagcc tcaaagtcag cactgtttct tgtggaacat gcaaaagcag 64320 cagagattatgacttctcca gtcctacaac cgaggtctct ctgcttcctg tgttcccgct 64380 acagaaccacgaagctgccc tgagacgctc actcacgctg tggggatgtc agagagcccc 64440 atcggacccaaatccacgat gctccgggct gatgcgtcct cgacgccctc ctttcagcag 64500 gcttttgcttcttcctgcac catttccagc aacggccctg ggcagaggag agagaggtaa 64560 gaagatggatctttaaactt ggcacaagtt ccccttaaag acccagaatg ctgagaggca 64620 tgtgggaccagcgggggctt gctcttcaga gaagctgaag ctagaccagg cagggactgg 64680 cctgaagatggggcggggca ggggaccagg gctgtgtgca cgacactgct accgccgggg 64740 acaggtgtatgtggtgacat acgagaaaat atagggacat acaggaaata caggaaaaat 64800 cagtcttagagggcttctgg tttacagaaa gacctggggg tgggagaggg tgggtattaa 64860 cagggaaaaatggcctattt ctgcttcaca aagatactac ccctgaggac attcatacgt 64920 gagaccattccacttgctca gagtaggaat aagtttcttt atgctttgtt gtatgaacac 64980 gtagctccctatttaacttt ttaaattcta cctctgttct ccttaaaata aaagaggagc 65040 cctcatttaaggacattacg catagactct gtacccatga atgttccatt gctagtgaaa 65100 tacgtgtgtcacgtggtgtg gttttagctg tctttccgta ctcgtaagtg gttggtaatt 65160 tccagtagtctttgtggtat ccgttaatca actggttcat ctagcaagca tggattgagg 65220 actgatggtgtatctggcag ataaaggaga tggggcagag ggatcatgct gcgaggagga 65280 ggggacccacagtgaaccaa cggcctggct gcgggggatg aatgtgggtg gcatccctgc 65340 ctagtgctgtgggacaagac gggagggcat ttctactccc gagggcacac gtgtatgtgg 65400 ccacaggtgctggctaaggg ctaggaaaag gcttcccagt gggtgcacag ctgagctgcg 65460 tcttaaaggacgaaagggcc ttgacccggt gaagatgcag gagggaaggc gcaggggccg 65520 agggagcagcatctgcgcat gtggaggcct tcatggggcg gctcccctgg gtgccctcat 65580 gcgggctgcagcatccaggc atgaaatggg ggcttgaaag gcaggcagcg tgagctcaga 65640 gagggccttagtgtacatat tttggaaaat acctcctgtg ctgcctggag ggcctcagca 65700 cccacttccccatccctgtc ttcccgtgtg ggtgacgctt gctggtcttc aggtgtctct 65760 ttgtaccttacatgctgtct tggtcggctt gtcttttgaa cagctcctct tctgcagaac 65820 gccagtgggtggagagcagc cccaagccca tggtttccct gctggggagc ggccggccca 65880 ccggaagtcccctcagcgct gagttctccg gtaccaggaa ggactcccca gtgctgtcct 65940 gcttcccgccgtcagagctc caggctcctt tccacagcca tgagctgtcc ctagcagagc 66000 caccggactccctggcgcct cccagcagcc aggccttcct gggcttcggc accgccccag 66060 tgggaagtggccttccgccc gaggaggacc tgggggcctt gctggccaat tctcatggag 66120 cgtcaccgacccccagcatc ccgctgacag cgacaggggc tgccgacaat ggcttcctgt 66180 cccacaactttctcacggtg gcgcctggac acagcagcca ccacagtcca ggcctgcagg 66240 gccagggtgtgaccctgccc gggcagccac ccctccctga gaagaagcgg gcctcggagg 66300 gggatcgttctttgggctca gtctctccct cctccagtgg cttctccagc ccgcacagcg 66360 ggagcaccatcagtatcccc ttcccaaatg tccttcccga cttttccaag gcttcagaag 66420 cggcctcacctctgccaggt aggtgtgttt ccagctggtt ggaagggccc ctctgtccat 66480 tcactggggaagggaagggt tcagacctgc agctgagctg gcatatctct agtgctgggc 66540 tgggcacctggagaggaaat ccagagccgt tggggtactt ttccattttc ctcgtccaaa 66600 accaagcactggctcaggct tttcattcac tcttcttggc ttctgtgttt ggccaccttg 66660 gattccttccagttgtagaa agtgcagaca tttgcagtgg ttctactttc catgtcactt 66720 gggaacatggctttttgtaa ataacaaatt ccttgtcaaa ttaaattaat tcatccaaaa 66780 cgtcattatgaaatatttca agtgtataag aagcagtgtg gtctatgggt tgaacaacac 66840 caaccttaccctctctttcc tcactacctt cctatgtatt cttaaaatgt attgcaaagg 66900 ctcaatccaagcatcctgtt gttctgtttt gtgttataaa acagtcactg cctcctccaa 66960 ctcagtcatttcaaaggcag ggttcccttt ccttagaaca tcgctcgctt gcttgccctt 67020 ggcatgggatttggacttct gggatcacag cagatgagag gttgcaagaa gaaaagtgcc 67080 catcatggtcactgttttta aattttgctt tacagatagt ccaggtgata aacttgtgat 67140 cgtgaaatttgttcaagaca cttccaagtt ctggtacaag gcggatattt caagagaaca 67200 aggtatgtggggaggatgag ggcctccttg ggcatctgcc ctagtgcccg cttcgctgcc 67260 ttgtgtgaagatgggccctg cggtgggaac atcgggcagc cgtggccttc atatggattg 67320 agtttgagcagggcccacac gttattccat gcctcctggg gacatgctac aacctttaag 67380 tttttataggggaaaactca ctactaatga gggagttggt gaggaatggg ccccagccat 67440 gagcattccagtgtgtaggg tgtaatttct ggctgcttgc accagccact ggcttgccag 67500 tgatgccagcccacaactgc tgactgtgag gaggagacac actctcctgc atctccatga 67560 aaccagagaggacagggtga ggaagcagga gagacaccct cctctgagaa gtcagggaca 67620 ctgtgtgtttgtgaacacct acataagcac ttaataatga gacccggttg tggacttagc 67680 cgcatgcctcacacacaaac cagacagagc atctctctgc ttggtacaat gtcaggcttt 67740 ttttttttttttttctaatt tgaaatcatt aatactaatt tttgagcaat gaatatttga 67800 atattttgagcatcactttt taccagagca tgaatagaag agatgtggga gaggaaggct 67860 agcaagctcttctgggcagc cctgcttgga gacagcccct gggcaatgag gagaattcat 67920 aaggtgcttccctcggctgt gggatggctt ttctccctgg atgctgtgtc tcagctgagc 67980 gtaccttcctctggatacct cattttggca gtgcatagag tttgttgctt ctgggatata 68040 cctcagaagagatggtgtgt gactggcaag gcagggcagg gccatgcctc cagtgcctgg 68100 aagacggagtcaggattcct aggaaccaaa gtgaatcccg gtggccacct ggtggaggct 68160 ggagtgaaagagggaatggg ttggtttaaa gcggtgagaa atgacaagct gcccgcagtc 68220 agcaggagacccacttggct cctctgtgcc tctttacctc aacttcagaa gaaaatcaag 68280 ctctctccttatgaagatgc ctcaggaatg cctagaaatg aatagttgag tggcagaaaa 68340 atagctctgtccagctctcc actgaaattc tgctattcag ttatgtagat gacagaaatg 68400 gtaagaatgtgcatctctta tgatctcaaa gaagcgattg tcattttaag tggaagagag 68460 agaaaaaaaagcctgtggcc tggaaagacc ttcatttccc tgaattggcc tttgtgaaac 68520 actgctcaccatggctgagg gcctctctgc ggccggatgc tgtgtgcctg ttgccaccac 68580 cctggccctgcttgtgtctc aagaatcccg atgactgtgg cccccagtgc ctcggcacct 68640 cctccagctggccaaactga gaggtgccta tccagcacct aagagattct gcacagaccc 68700 ttccactctattggtgccgt cttctagggc agggcgatgt gtggggctgg atgccccctt 68760 ctctcattgtgcaatgtcat tgtagcaggg gggatctgaa acagacagat ttgggttgga 68820 gtgcactgctggcttcctcg gcagggtagc aaggttcgag cctgccgcgg gggtttgctg 68880 agagcagtgccatgtggaag gtacctgccc ttgctggcgc ctggtagatg caggataagt 68940 ggatgtggatgtgcagtatg tgtaggggct gctgttggca tgtgcttgat cttcagcaca 69000 cattgtagaaggtttgctgg gaggacccca tctcaggatg gccaacatgt gcatcgagtg 69060 ttggtttttccagctgagct ctaaaaggat aactttcatt ttctacatat tttacataga 69120 agagacctgtgcagacgaac cttctactgc ccgtgtcaga gtctccaaac agcggaaggc 69180 tgcccttgggtgtcatttat ttggatttta actgtcagat gtgccttttt ctagaaaagc 69240 tacaatgacatttcagggtg atgcaggagg aaaaaaaaca cccagataac ctctttggga 69300 gtgcaatttgtcacatactt aattccttga ggtttcttaa agctaacata aaaatacctc 69360 aggaggaaaagaggacctta aagtaaaatc aaaattaagt actactgtcc cagttgtact 69420 tctggggtgttgcttctcca gatgcttctc cagatgcagg gcgcgacagt gtgagaaggt 69480 gcgaggagactcacttgcct cccctcccct catccctgtc tccaggtgag gaatgcgatg 69540 ccagtgccagggtggagcac gcagctgctg tgtgcagtgt gtggacgctc gtttttccaa 69600 cttcagcacaccaggctcct cagcctactt ctgtttggaa aaacagcggg tctaaaattc 69660 tcctcttactcccattgcct gtgcaggaga gcacagcccc cttgcacccc tgcagcctgg 69720 cagctggggcttccctcctg gcatggggag aactggtcgg agcaaagccc gtgttccctc 69780 cactgccatccacaggtgcc caaagtctca gtcccgtgag gcttctacac tttatttacc 69840 acagagaagtagaaccatga cttacaaaaa gaaaagctct gaggtatatc acagttgaaa 69900 caaaatgtaagtgatctttt gtcctactta gagaaaacaa aaaagtaggc ccggcgcagt 69960 gactcactcctataatccca gcaacttagg gaagctgagg tgggaggatc acttgagccc 70020 aggagtttaaggccagcctg ggcaacatag tgagacccag tgtctaccaa aaatttttaa 70080 aaattagctgggtgtggtgg tgtacacttg tagtcccagc tactcaggaa gctgaggtgg 70140 gaggatcacttgggcccagc aggttgaggc tgcagtgagc tatgattgtg tcactgtact 70200 tcagtctgggcaacagagtg agagagccta tctcaaaaaa ataaaaatta aaaactgaaa 70260 aaatgtcattcacgttcagc ccccaagttt atagactacg ctgtgagaaa acgtgcacca 70320 gaattcagggtttattccca gggcctgtgc cctgccccag gaggcccccc acccagccct 70380 gggtcccttgcatgggcggc tgcaggcctg gctggagcta ccagcagtct cgggcatgca 70440 cggtgagcctgcctgtgttt tcctgcccat ccttcccact gcgtgctgtt tagaagctcc 70500 tgcttcatccccaggggcat gttgagctcc tccccgtgcc ctcatcctgt gcacagggcc 70560 acggtgcagacattgaaaaa tgtgtaaaag aaaccattcg tttcatccta tggccagaca 70620 gatgccttggctgtgggtcc ggtcaggcct gggggcatgt gaggggcctc tgtgggagtg 70680 cgccgtggaggtatttactg aggcatgccg ggggcatccc agcacttact ggttcatgtt 70740 ctgcgccctgatcgcttcca ggcagagcct ttggcattcc agaaaggcag catttccatg 70800 gctgtaattatgtataactt tatactctaa atccccagtt ttcccttttt taaaataaag 70860 agtaaatgtctagaaaagaa attctgagat gtagtctagt tatcagggaa cactctggct 70920 tgtctgggcagtatcgagtt gaggttacta ttaccctaac ccactccctc ttcttggaaa 70980 tgccagattttggccatgaa ggagagagat tttttgaagc aatgattatg attagttgaa 71040 tgcctaccagcctcaccagg gaagctgggt gacccactgt gcgaggccct gggttggcgg 71100 gcacatggggaatgtgtcct cagccccatc caggctgggc ccatttggcc acatcagcca 71160 tggagccacgggataggggg ccacctgcag gccaggtttt tgttacttcc ttggtgaatc 71220 ttggaatttgtattaaattt tcagtaacat tttgggtatg ccctttctat catcagaaaa 71280 tgtaattagaaacttctttc ctggccaggc gcagtggctc acgcctgtaa tcccagcact 71340 ttgggaggctgaggcgggtg gatcacaagg tcaggagatc aagaccatcc tggctaacat 71400 ggtgaaaccccatctctact aaaaatacaa aaaattagcc agacgtggtg gcaggtgcct 71460 gtagtcccagctactccgga ggctgaggca ggagaatcgc ttgaacccag aagtggaggt 71520 tgcagtgagccgagaccatg ccactgcact ctagcctggg caacagagcg agactccatc 71580 tcaaaaaaaaaaaaaaaaaa aacttctttc cttttatgct gttgaaagag ttcatgttct 71640 catgttctaaaatctttaag aaaaatgcca cagggggcag tgtccaagca gaggcagggt 71700 gttctgcgacagcctccctc ccactgcctg tttctcccct ttgtgtatat ttggatctgg 71760 tcttgtttcttactcagcgt gagccttggt ttcctgggct gtcaggtggg gaccatcatt 71820 tggacatcacaggtattgtg ggattaacag aaatcctgtg ttcttccatg ggcagcccac 71880 aggacagtgcccggcacctc agtctagcta aagagaggga tgttcctgtc ctttctgcct 71940 cctcaacaggcaggcccacc catgaaacat acacgacacc acagagacct ccctgaaggt 72000 ccctcaactgcatggacatg tagttcttcc agccaagcag agggatcccg gccaggtccc 72060 cactgatccagtttgcaaaa agagtaaggg gagaggacct acccaaggtt atgagtgggc 72120 atagacctttgtccacccgg cagaccttct tgtctttctc ctccctttct ccagccatcg 72180 ccatgttgaaggacaaggag ccgggctcat tcattgttcg agacagccat tccttccgag 72240 gggcctatggcctggccatg aaggtggcca cgcccccacc ttcagtcctg cagctgaaca 72300 agaaaggtaaggttactgct ccctattgat ctgttcagct catccacaga gaaactctgt 72360 tggtttatttttaggctaat aaaataaaag atgactttaa caatactcac aaattttatt 72420 cttagtatttattttttcag gtatatttta caaagttttc accttattta agaaagaaca 72480 ttaatttgttaattttattt tctattgttt tgaataggta atccatttca ttgttcaaaa 72540 attagaaccatgggataagg catccactaa gcccgttttg cttccatttc ggctctaccc 72600 ccctcaccaccctgtcacct cccttgggca accatctgga gtagattcgt gtgtgtcttt 72660 tcactggttacctgtccaaa tgcaggcaaa cacatttttt atatgaaaac tagcatactg 72720 ttgagtactattcttgtatc tatacccagt agggcatttt tagatggatt aaaagtgata 72780 ctagaataagcctagactca tgagatgatc tctaatgcag gtaaaggact atgcctgcct 72840 gaatcctttttgcattaaaa atcatggcaa aaaccataat gacttttgca ccaacctgta 72900 atagaaggatttctattcct ttgggtataa tacccagtaa tgggattgct gggtcctatg 72960 gtattactgcctctaggtct ttgaggaatc gccacgctgt cttccacaat ggctgaacta 73020 ttttacactctcaccaacag tgcaaaagca ttcctttttc tctacaacct caccagcatc 73080 caccaatcatatttctaaga ttgcttgtag ctgttgttct ctttggaagt tctggctttc 73140 tatcttatgaaaatcctaca gccaattgat ccattctatt gttggtggac acttgatttt 73200 cagttttcttttttttttct ccccctgccc tttttgcccc attatactca tctttgtgtg 73260 ctaccatgctgcagactaga ctaaggaaag ggaagctatt ctttcactct gtctgtctcc 73320 ccaaatggaaattgtggact gaggggacat tgcaggtcgt tggttcccag taaggacagc 73380 ttgattcactaggagtgggg aaatcagaag cagctcggat tttaaactcc ctgtgtttat 73440 agaaaagtgagacacccacc tggttcactc atgctctgtg cctaatacct gagagaattt 73500 ttccaaattcttggatgaaa cctctgcttc atgcttcctg gagaaaatcg ccttggagtt 73560 acaccctgtggaatgtatga tttggctgcc aggaggtcca gcctcaggca gggctgtagc 73620 gtttccgggaaacatagatg ggttcatttt ggcacaggta atggtgtgag agtggaaggg 73680 gtggggagtgaggctgggaa agacccgggg ctcagtcaaa gctcagcccc gaagcctgtg 73740 gctactgattgtcctgaagt gaagtagaga gaaggcgttg tgtaggggag aaaggacaga 73800 taattcttgaaaagtaggat cttctagcat ccagaattcc agcataagaa gcccaacacg 73860 atgctatttctgagtggaga taagggcttt tgtagctgga gagctgaatt agacctgatt 73920 gttgaggactgaaactgaat aatgatgatg gcaggaaaga gggtcgcagg accaagagca 73980 tctggcgagtctgcagagat gagagattat tgggagagga ggaatgatgg atggttttgg 74040 ttggctccttcgtttccgtg taaagtttta aagcaaggac aaataacatt tctgcatgca 74100 ttatctttttggaatctaga tgagagtata taactgtttt tacctctgtg cgtgcatcct 74160 gcctctctgacagactttgg actcaatctt aagatgaaag gaacaaaata ggggatatta 74220 gaatagcagacttgaggaaa ccgagtgttg ttggtggcct ctgggtgaaa tcttatctga 74280 ctgctgtgtcttcatcttgt ccctggattt gacattcaga tttcacaatc caaggataga 74340 tttgagcaaagagtttacca ggtcataaag agccacaacc attcaccata ccacctccag 74400 taaactggagatgggacagc tagggacgtt ctggatgctg ttctccagag agccctggcc 74460 agaaggacttcccactgagc catttctcac aagtggaatc cagacccacg cttggtgtcc 74520 ccttcagtttggaagccagg cgtcattttc ttcccacaac tgaatgtcaa tatgttctta 74580 tcccactttaattcaacaca gccttttatg gaatctgatc atttcctttt ctgcaagctt 74640 tactgcccttgtgaaatgct gacacagtga tatttgtcat gtttataaac atcttttgga 74700 agcctactgtgggcaaggca ctggggacca agcccaactc cagtggatct tgctgccagg 74760 aggtttccaacaccagaaat attagaagtt taggcagaca tgccccaatg gagggtgatg 74820 aaggaagcactggatcccag aaatacagca cactgtgaag gtctgttgga ctcattagca 74880 tagggcagtgacttcttgtt cttttggttg tgctgtttgt acttttccac tctcagatag 74940 ttttgccattcagtggcttt ttatgatgtg ttttctcttt ggactcatgg gctggcctaa 75000 agcagatgcactgtttactg ttaatagcaa tgtggtagag tgctgctccc gtgcagctga 75060 gtgcttgctttctggaaggt tttaaagatc caggctcctg gttaaatccc acccagaaga 75120 ctgacctccttccagctctt ttcagtgatt cattataggt atcattttgt gtctgagcct 75180 ttctcacctctcctctttgg gagaggattc agctggagat tcatgatcct ctttcttagc 75240 tgcaagctcattcccttcgt tggggattct tgcacaatgt ttgagggcct ctcccaaggg 75300 acctctccagcccccacact gtcattgcaa gggttcccgc cctgtatctg gcctagcctg 75360 tgctcatccagctgtcctgc agcccggcta ccattcctag gacgaagatg gttcttggca 75420 gcctcttttgcagtgactgg actcgtattc agagaagagc cttttcaact gtagctcctg 75480 gcttttatgttctctctatt gaattctccc atgctcccat tttcaaaaaa ttcttctttc 75540 tttaaatttcttgccacagc tggagatttg gccaatgaac tcgtccggca ctttttgatc 75600 gagtgtaccccgaagggagt gcggttgaaa gggtgctcga atgaaccata tttcggtgag 75660 ttgcttgagaggttgctgtc aatgttctga actcatgagg cctggattcc ctctgctcac 75720 caacctgagatttgggacta tcactcttat ggtggtttac tcatatttca gtccccagtt 75780 ctgaaatcctgctgatctgc attattatct ttaagataaa ctagatttga agttgttctt 75840 aaaagctctgtcaaaaccaa acaaatgaaa agcaccatat cagacgtagg aattacagac 75900 gtcaataagattctatccta gttggcaggc aggtaagtcc tgagaaggtt ggagctccag 75960 catgaccctatcccacagca caaggaacaa catagagctc ccaacagagc accctccctt 76020 ccctgcaaattgggagcctt ccctacctca cgagcttgtt ggaaagatga cttgagcaat 76080 ccacacaaagcacagtgcag ccttaggcat cattgttatt agagctttgt taagaagcat 76140 tgtgcaggggtaatatatat ggttgaagtt ggagttggct tgatgtcaac cctcctgatt 76200 caagatccaacaaactgtat gctttagtct ttatataatt tggtaatgat gtgtctatga 76260 taagaatgataaacagttcc atttacttac ctctcaacta tcttgtggaa ctttatactt 76320 ctttgaagttttacacaaag ctgcacacta atctaacata ccagttgcta aaaagataag 76380 ggctcagcctttgcatccgt ttcattccag ctgataaatt ggctgagcac acaacatcta 76440 cagcagttgttgactcttgt ttgctgtctg tccactttgc agggagcctg acggccttgg 76500 tgtgccagcattccatcacg cccttggcct tgccgtgcaa gctgcttatc ccagagagag 76560 gtaaggggtcctcagtgcta gtcaggctct gtgtagattg tctgcagtca aggccaccgg 76620 ttttagagatccatgaagat ccactgagaa aaataataag actggaagtt ttttcgtatt 76680 ctcttggttgaacccaagaa aaggccaatg agttcatcca aattaaggag agaagaggag 76740 attctacttcactgacagct cccagatagc tttacatcat ggattttgat gaaagatttc 76800 tatagagcaaagattctgta gtttaaaaat ataaaaccct cagacaaaga aggcttcagg 76860 gtcattgacatttaattttt acgtttctac ggaatgggca aaatgttgat gttgttgaac 76920 ttagatgatggatgcactgt tctttttact tttatgtttg ttaaacaata tccataatga 76980 aattaaataacacccacagc atgtcagggc gatgtgaccg tatgtcccac tttcctaagc 77040 agtcacaacttcacataaaa atggtctaac ttaatatata ggtaaatgtg atttactttt 77100 caaagtgtcattgttcatat gactaattcg gtgattagaa aaatattttg ttaggccttg 77160 tcatctctacttttattcag aatgtgatca tggtttctat gggccaaatt attgtcccaa 77220 agagaaactcttgactctgc aaaaccattg catgtcacca tctgggaatt ctgtcatcaa 77280 attgtctaactttgtcatga ggattcttgg tcaggagcag gactcatttc tgtattttct 77340 tgccccttcctacagatcca ttggaggaaa tagcagaaag ttctccccag acggcagcca 77400 attcagcagctgagctgttg aagcaggggg caggtcagta aatgcagctc catttctaca 77460 tctggtgactggggagaaaa aggactcact gacacaactt ccttttgcac ttcagtattc 77520 aggtttcctttcttttctgt gagccttgaa ggaggtttcc ttttatagaa gcttgcacag 77580 agctgggtggggtgcagttg ttgggacatc agcaaggcct tcatgggccg gtctaactca 77640 agcaagactaaattcagtgg aataaatgtc aagccctgga tttagtttgg gggaaaatat 77700 agtaagacaggatgtgggtg atctgtctcg gcaactgtaa atgtaaaaat gttccagaga 77760 catgactcaacaagtcactt gacatgagtc aatgatttga cccacactgc cagcaacccc 77820 agtgagacatacaacaccca agaaccagct cgccagacag agcaggtcac ttaaaaaaat 77880 tattcatagagtttcttttt tagagcagtg acagattcac agcaaaatgg agaagaaggt 77940 acagagatacaccatataca cgcatagcct ccccgctgtc aacatccccc agcaaagtga 78000 tgcacatgttatggttgatg aacctgcatt aacacagcct cgtcacctga gtccctagtt 78060 tagggacttgatatttaggt tcaccttgat gtcatgcatt ctacaggttt ggacaaatgc 78120 ataatgacaggtaatcaccg ttacagtatc atagtgaata gttttattgc cctaaaaatc 78180 ctctgtgccttttctgttcg ttctccctcc cccaccaacc ccaagcaacc actcatattt 78240 ttattgtctccatattttca acttttccag tattgtcata tagttgaaac catacagttc 78300 atagccttttgaggttggct cctttcactt agtcatatgc atttaagttt cctccatgtg 78360 tttttatggcttgatagctc attttttttt agcagcaaat atttccttgt ctgggtgtat 78420 cagtttagtaacccattcac ctactaaagg acattgtggg tgctttcagg ttttggcagt 78480 aatgaataatgctgctctaa acacccatgt gcaggttttt gagtggatat aagttttcaa 78540 ctcctgtgcgtagataccaa ggagcgcatt cctggattgt atggtaagag tgtgtttggt 78600 tttataaggtacaggcaaac tgtcttccaa tgtggctgtc ccattttgca ttcccactag 78660 caatgaatgtgagttcttgt tgccccacat cctctccaga atttggtgtt gccagtgttc 78720 tggaatgtggcctttctaat agtagcgtag tggtatctca ttgtttctga tgacttatga 78780 tgtgtagcaccttttcatat gcttatttac catctgtata tcttctttgg tgaggtgtcc 78840 ataaaggcctttggcttgtt tgtttaattg ggttttgttt tcttattgtt aaattttggg 78900 aattcttggtgtattttaga cactagtcct ttacctgata ggccttttga aaatattttc 78960 ttccatttcatggcttgcct tttcattctt ctacagagca gaagttttta attttaataa 79020 aatccagcttattgatttcc tgttttatgg attgtgactt tggtattgta tcttaaaagt 79080 caccaccaaacccaaggcca gctagatttt atagtttcaa attttacctt taagtctgtg 79140 atccgttttaagttaatttt tgtgaaggaa gtaaggtctg tgtcttgatt cattttttat 79200 ttttttgcctgcagatgtac agttgcttct gtaccaccag ttgaaaaatc tgtcttttca 79260 cagttgtattccctttgctc ttttgtctaa gatcagttaa ctatattcat gtgggtctac 79320 tttcgagttctttaattgac ctatttgttc tttcaccaat atcacatttt cttgattgct 79380 ttagctttatagtaatcctg gaagtcaaat aatgtcagtc ctccaacttt gctcttctct 79440 ttcaatattgtgtttgctat ttttgatctt ttgcctttct gtattaactg tagaatcact 79500 ttgttaatatccacaaaata agttgctggg atttttattg agattgcatt gaatctatag 79560 atgaaattaggaagaactga caccttggca aaagttgagt cctgctgtcc atgaatatgg 79620 aatatctctccatttattta gttttttgta tctttcatca aagtttaata gtgttcctca 79680 tatagatcttgtctatagtt tgttagattt atatgtaagt agtttttggg aggtgctact 79740 gtaaatggtattgtgtattt aatttcaaat tttacttgtt tattgctggt ctgtaggaaa 79800 gcagttgaccatttgtacat taaccttgta tccagcatct ttctgtaatc gcttactagt 79860 tccaggccaggttacttttg cagcattaga tgcagtcgta gtggccccag tttaagaagg 79920 ttgtaaacaaaccacacacc atagggctct tgtggttctg tggccatgag ggagggacac 79980 agctagagaggacagtgggg actgctgact ttcaaaccct gactttcaaa actgttttaa 80040 gtttttcatgcaaggtaggt gtttattgtc agaaatgttg gggagggaat ttctgagtct 80100 ggtagaaaattggactagaa atacctccat tctgtacctc atatctgcag agtagtttac 80160 aggtgtaactccacaatgaa ggtgtatgaa atgtataggc attttcttat ttcctctaca 80220 atttgtattattcttctatg tagcttttcc ctgtaaaatt atacatcact gtgtctgtta 80280 ccaagctaaagtaaaattac tctgttttga atggaatttt gtagtgttta agttatttta 80340 gactctccttataggataag ttatctgtgg ccaaaacaaa ggtgaaaata gcttgcccca 80400 aattgcaagtgtgctaaaac tcgaagctcc aaaatgaaaa atgtagctgt ggtcatgggt 80460 ttccggccatgttctgctac tcaggatttt gatctctggc aaatcactta gccatgcgga 80520 cctgaggtttgtatctggtc agtggggata ataatatttc cacacatagc agttgaaaag 80580 attaaatgaataggacattg acaatgcctt gggagcattt ttctggccca agaggaatgg 80640 cctttcctgggaagccggcc tcaccctttc ccttgtgagg gacctgggct ggtcctgggc 80700 cacagcccctaaatctgttt tagggggtcc ctggagccca tcatggttct tagctttctt 80760 ctgttaaagtaacagtggac cctgtctgtg ggtcccagtg aagtgcccag aaggatgcag 80820 gaatttactgtagacctatc tgatgagcag ctcactcact ctcaactgag gagacagggc 80880 tacaaaaccagctccactct gatggattta tttgtttcag ttgcaccttt taaaaatgta 80940 tttgaaattttgaaaacctt taattagtgg ttttaaaatc acaacttatg catcggcagt 81000 gttcaagaaagcgcagaaga acggtgatag caacaggcac agagcacttg tcctgcccag 81060 cttattgtctcctccccttc acccacaggg cgtgtgggtg tagtgtcctg tcatcccatg 81120 tcatagacagaggacccaag ttggcagctc tggctcctga atggtagatc taggcctggc 81180 attcagctcccacgtccagc tgtgcccata ttgtctcagg gacacagccc tgcccattca 81240 gccctatatggtagcaccag ggcacacgca atttcacttt tctccttaag cttcttagtt 81300 atcctgcattcacccagtaa gtgtggaatt aataaatgaa ttgaatgaat ttagttgatc 81360 ataacagtgcatcctaatag cacttgtttt tagatattaa aactttctct tctatcttga 81420 cttatcatttttaagtgcac ttccatcttt taaaattacg tatgaaatgt atcaataact 81480 ccaaatgttttttaagggtg ggtcttctta gagtcaagac ttcattcaga acttggagtt 81540 tgtatctgatcatctgagtt gggcatttat atatgaagct taaattatga ctgccttgag 81600 cttctttcctttaaaaatct catttctgaa acacattgag agctgcgaat gtctgctgtc 81660 cattacattaaccgcgattc caagccacgg aaccaggaca gcagatctgt caccagagga 81720 gtgaggctgtcacagtgacc acccctcagc tagtgtcgtc ttcacctgga ggattggctg 81780 caggtgtttagaatcagtgc agaacaacca ttgtagcaag acggtggaat tattgcaatc 81840 tctgatgacctccagcaatt tagtgagttt tctttcctaa actgaacttc atctgttgtc 81900 ctaattcacttagagtccta gactcctgga tgtgcaatgt tctttgagat gatcatgtcc 81960 aatttccccgttataaagat tcaggaagct gaaactcaga aatattagca gacaactcac 82020 aaagatgggaataaacttgt aatgctctcc agtttaatcc ctttttttaa atattaaggc 82080 cattatgcctttgttccaat ctctaaaaca atttctattt gtgttttaag cctcggttct 82140 taattgtttcttgttgaagc agaggaatat aatataatcc tgaaaagaaa gacttacttc 82200 ttttcagtagccttctaata gatttcccag aaatgttgca gttgcatttt ttagcatatg 82260 gtaggtatactttttttttt ttatagagtc ttatatgttc tttaagtttt aaaaatggaa 82320 aataaaagaaaaggaaaaat gaactactca tagatcggtt gcccctatgc agcccctgaa 82380 tattgggaggaagagtggac agcaggtaaa gtgttctcca gcccaaagac cactcctgat 82440 gtcaagcagatccaatccta tgctgccttt ttccttgtac ttggggtcat gctgcctgta 82500 cgattcttttctcttctctt agcataataa gtttacccat ggtttttaca aacataatgc 82560 aggtctcatttttttcatgg ctacacaata gtccatcata taattcacct ccttatggat 82620 gttcatttaccatgtttcta atttttaata ccacagacaa tgcttagagg accatctttc 82680 tgcattatttattttttctg aaatctggat tgttgcctta ggacaaattc ccacatgtgg 82740 gcctcagacttttgttctgt gttatcaagt ccctttccca gagtagccta gcaattgaca 82800 gaactccatgccatgggtct ggtgggtgcc ttcctggtaa ctcttactgg gcactgaatc 82860 ttcacctctggccaagagca cctgtcaagt ggcattggat ggtcttgggt caggtgcagt 82920 tggacaatcataggcaggcg caggcatgcc tgttctctgc tggagagagg ctttggggca 82980 cttggtgtcagttggttgat gtgtgctccg tagaacagcc tccctagtgg cagcattttc 83040 ttctctgctttaaggaatga tgacatagtt cctgcatttg gggagagaga ttccccaaac 83100 agcagattgctaccaaactc tagccagcaa gactgctcct catcagaaag ccatctgtga 83160 ctccttcaagagagcatggg tctgggttgt ggcagctgtg gttctgtgtg aactgattga 83220 gattgtgccaattttgtgga attttcctag tagttaggtc aaaattttcc cttttttctc 83280 tctttcataactgcatgatc agcatgacag taatgctaat aaaatcaaag gcggactgtc 83340 cctctcgtgaagtctgtttt gaattctcca ggctccctcg cagctcctat ctgatgcaca 83400 catgcttttcattttcccgt tctaatcata gcagacgtgg tttttattct gctttccctg 83460 ctgattgctgatgacgtctt tccatgctgc catgtgattt tcctggttac cgcttgctgg 83520 ttgcttggtattacaccgag ggagctaatt tcacccctct ctcatttcta atgtgtttct 83580 ggtttttcccactgtaatga atatgtggca caaatgtctt tccctgaagg ttagctatcc 83640 tatgtggttacagaatattt cctttctcaa tctcctcaga agcgggacca ctaggttact 83700 tctaggcatttcatatccac ctgataaata tcgccaagta tactagtcgg ctctcatgat 83760 gctaatacagccatatctga gactgggtaa tttataaaga agaagaggtt taatggactc 83820 acagtttcacatggctgggg aggcctcatg atcatggtag aaggcaaagg aagagcaaag 83880 gcacatcttacatggtggca ggcaagagag tatgtgcagg ggaactaccc tttataaaat 83940 catcagatctcgtgagactt actcactatt acgagaacag catgggaaaa acccaccccc 84000 atgattcaattacctcccac caggttgctc ccaccacaca tgggaattat gggagctaca 84060 attcaagatgagatttgggt ggggacagag ccaaaccata tcaccaagtg atcatcaaaa 84120 atagtgggctcaattcctca gagtgtcttc agctataaac aatatggcct ttgcggtgga 84180 tagaaaggatggtccagcaa ctcacaagtc accctgcact gtgacaccca gggacatcct 84240 agcccatctgtgggcttgtg tgtcaatgca ccctgtggca cacctgtcag accacctgcc 84300 agcacaccttccagtgcacc tgccaacaca tctgccagaa cgtctaccaa catacctgcc 84360 aaaccacctgccaacacatc tgtctgtgca cctgccaaca cacctgccaa taggcatggc 84420 ccttgctggttctgaaaagt cagacttaaa ggcaaagaga aaatggagct gcttcccagg 84480 gctgcctaccccaggaggac acggccgagg gagacaacac tacccagcgc gtaccgtgca 84540 tccgagtgctgagatgtgtt gctgtagact gtgggaaaac actctttttc tgttaagtga 84600 aactttagaggcctttaacc acggaattaa gacacccaca tgctcacata gagggacatg 84660 tatacagtagacaaggggca agtgagtgag ggcagttctg ttcctcagga ggaagcctac 84720 tcagcctttgggcaggagag gccctggttt acttcagtga atgagcttca ggctaaggct 84780 ggatgtcttggaacacctca atttcaccca tgccccaaag tcacacgtgc acttatgctt 84840 gaactgcagggtcacaggct agaagggaaa gaagggttgt tatggagccc agttgttttt 84900 cttctcccaagaggagatgg tcagggacaa gacccacagc tctcccttgg cagcatgggg 84960 agccaggacacaggtgaggc cgggccactt ttggcttttt ccagcctgtt ctctgagctc 85020 actgcaagccattccttctc ttggcaacca ggatttgagg acacatgctt ccctcatggt 85080 tgtttctcaagggcctctgg gctccttacc tataacagta gttgggtttc atattaatgt 85140 gactgttgtccatacccacc ctgagaccag agaaggtgcc ttgggctcca cctgggacac 85200 gagggtttaatactgctgca gggcgaatag aaagctccct ctggctggtg ggaaggcaaa 85260 gccacaccaggcgggtctgc tttgctctag taactcgctt attctctgcc ttgacatggg 85320 agtaagtgcatccttggagc actcactgtt catgttttca gaattagggg caaaggccat 85380 ctgccttacatgcacccagc ataggccaca tgcgcaagtt gttcataaac ccagtctgca 85440 catcagcaactgcctgtgtg ttaacaggac acacctgtcc caatagttgc aactaacatg 85500 tgtctcccttggtgtcttcc agcctgcaat gtgtggtact tgaactctgt ggagatggag 85560 tccctcaccggccaccaggc gatccagaag gccctgagca tcaccctggt ccaggagcct 85620 ccacctgtgtccacagttgt gcacttcaag gtgtcagccc agggcatcac cctgacagac 85680 aatcagaggaagtgagtgcc tagagggagg caggagccgt ccagcagggg cggggctgtc 85740 acgttcctcttgttcttccc tagtgtgagt agtcatccag gtaaatcatt agccgtcttt 85800 gaggtcatgagtcacccagg gtctgtctag gtatttttct gtcattaaat acgtcctggg 85860 aaccacataggaactcttag catttgtttg caaggctatt tttcttagaa atcagtaaaa 85920 taaaataaaaaaagacgttt ggcgttttaa aattacacac atacacacat aattgcaaat 85980 gcaaaaaatcttttaactca gttccttaaa aacactctga tactatgatc agagtccaat 86040 gatcttggaacctattgttt taacagaggg tcatttcctt tcaggtgagc gtatgacctt 86100 gatatgtactatttccgttg gccaatttca aatgacttac gcctgagctg ttactgagcc 86160 agttatgctggagcagatgt tctccatgtg gatctttact gaatgcccag cataagcatg 86220 gaacagagcagccacagggc tgttcctgtc tccctccctc cagaatgcca tccctgagtg 86280 cagcctgtggcctaggaaca gagcatttca gatgcaatta ttttgtattg tgtggttgag 86340 gaggcacgttggcatcaacc gtggcagcag catgatggca gcagccatca cagcagacag 86400 gccggcaggggacactcaga ccagcctggc tgctttccca ggatgctccc cgagcagggc 86460 ccccatgcctctctgtggtg cccatgtccc tagaagcaag acattttctt catctgccca 86520 aaccacctgggaggttgaag gcaggatctt cccctctgta gcttcaggaa taagtgagtt 86580 taggcaggtttagctcatca cccttcatct gctgaaaaaa atgagcacaa agcagaacgt 86640 gggttttgctggtcagaccc gttcagtgcc cctgacactg accagtagat gaccctgggc 86700 agggtcccaatgcttgatgc tctcgtattt caatcctgca ccaaaatacc tatttcaagg 86760 tttagtgtctcaaatgaaaa gggcactcag caagtgccct gtgtggcaca cgcagggaca 86820 gggtcagggttgtggctcaa ccccaccatg ttctcagggt caggggcatg gctcagcccc 86880 actgtgttctcagggtcagg gtcacaggca aggctcagtt tcacggtgga ctcagggtca 86940 gggacatagctcagctgtat ggtggactca tggtcaggtt cagggggcat agctttgctg 87000 tttggtggactcagggtcag agtcagggtt gtgccttggc tttatggtag actcagggtc 87060 ggggcatggctcatctgcat ggtagactca gggtcagggg catggctcag ctgcatggct 87120 cacccagggtcccagttggg tatggctcag ctgcatggtg gactcagggt cagtgtcagg 87180 catagctcagttacatggtg gacacagggt caggggcatg gctcggctac atggctcact 87240 cagggttcccgttgggtgtg gctcagctgc acggtggact cagagtcagt gtcaggggca 87300 tagcggagctacatggtgga cttagggttt gggatatgga tcatctacct ggtgggctca 87360 gggtcaggctcagggcatgg ccaggctgct tggtggactc aggcttacag tcaggagtat 87420 ggctagactgcctagtggac tcagggtcag ggtcagggtc aggggcatgg ctaggctgca 87480 tggtggactcagggtctgga ggcatagctt ggcgacatgg tagactcagg gtcagggttg 87540 ggctccttaaggaccctcac aatgttgact gttccctcct ccctgctgcc cttggaagat 87600 atgttatttcttcacaggat ctgttctatg tgcccttgtc agagttgtct tttttctgtg 87660 tctctttagtttccctgtat tctttatatt ccctggagat gggtcatgtc cacatcagtt 87720 cagatctttcatactgtaag agatagcgtt tgagttcaga ctggctaatt ctagacaaaa 87780 gggggatcttctcaccaaca atcagcctta aaaggcacct gtttgagagg ggctgcaggt 87840 ggctgcagggtcaccttgaa ttgaagcaca cccaggctct gcctctgccc tagcctatct 87900 cctggcctctctcttcatct ccagatgagc tttactctgt gccggtgcag agtcattccc 87960 agaaggagaggggtgacccc aaagccagct agactggccc agcctggaca cagcagagtg 88020 ttgagttgggcatcctctgg ggaccatgtg gactgggtgg atagcactct tggaaggaag 88080 ggctgggcaggtgagccgga ggagaatgcc ccaggtggtc atgtcagtga ctaagggctg 88140 tcaggtgagcctggaaggag gtctctatgg gggtcgtatc agtgaccatg tggagtcctt 88200 ccttaaggaatattccttgc acagcacttt attgttcact cagtgctctc agacctgtga 88260 ctgcatctgacttgggcatc tgtcctgtgg aaggggtggg ctggtaacat gatccacttt 88320 tcacaggtgaggaaaccaag gctcggagcc tggaggctgt tagtggtttc cccacgatcc 88380 tgcagccagcagtgtctggc agggccagag gtgcttcctt gtgtccatct cacaccctac 88440 cagcttttccagtttgttag ggcttttttt tttttgagat ggaatctcac tctgtcgccc 88500 aagctggagtgcagtggagc gatctcagcc cactgcaacc tctgcctccc aggttcaagc 88560 aattctcctgtttccacctc ccaagtagct ggaattacag gcacatgtca ccacacccga 88620 ctaatttttgtgtttttagt agagacgggg tttcacatgt tggtcaggct ggtcttgaac 88680 tcctgacctcaggtgatcaa cccgcctcag cctcccaaag tgctgggatt acaggcgtga 88740 gccactgcacccggcttgtt agggcttctt gacccgattc ccgccttcca cacggctgac 88800 agtttcagcctgtgtggtgc ttccgtctac tggtaacaac tgttctgccc agcttcatga 88860 catctgttgactttgaatga gcacgcactt tcttctctcc aagtgatgag taaaagtatg 88920 aaatgttttctgctggaaga atcttcatgt atgggagagc ccgcccagtg catcccccat 88980 cacccaaaagccctagtaac aggagccatc ctctctcccc catgtcccaa caggctcttc 89040 ttccggaggcattaccccgt gaacagtgtg attttctgtg ccttggaccc acaagacagg 89100 aagtaagaaatttgcatttt tattgagcaa ggagtgtact taattctatc ctttagttta 89160 aaaaaattaacctctgtttt tctctttaat ttgcaaggtg gatcaaagat ggcccttcct 89220 caaagtaagttgctgagatt tctttacatt ctctccttgt ctgcagttgt actccaaagt 89280 tgaattctcttccagatttg cacagggggt gccttataag aaaatgacta caccatgcca 89340 atttcttttaaaaaagaaaa aagtaaaagt gttctcatta tggacaagga tctggatgct 89400 gttaacttttctgccggctg ttggctggat gtgcaatatt tacatgtgtc atataacact 89460 gtggcatagatacagggaga tggatggcac agcctttttg gccatttggg aatgcatggg 89520 ctcagcagtgctgaggtctg cacagctaga aggaattgct ccctttgtgc aggtgtgtgg 89580 atgaagcgggtgttccaaat ttaccaaatg caacggagga gggccttcat cacaccgttg 89640 ctccacagccagtccaggca gctgttagag tttttttttt tttttgagac agagttttgc 89700 tctttttgcccaggctggag tgcagtggcg cgatcttggc tcactgcaac ctctgcctcc 89760 cgggttcaagcaattctcct gcctcagcct cccaaatagc tgggattaca ggcgtccgcc 89820 accaggcccagctaattttt tgtatttagt agagacgggg tttcaccatg ttggtcaggc 89880 tggtctcgaactcctgacct caggtgatcc acctgcctca gcctcccaaa gtgccgggat 89940 taaaggtgtgagccaccgtg cccggcttag agttcccctt ttataagcac acctttccct 90000 ggctccctgagtaccgtggc cttgcttggc ctgggctcat tcctggccca gtgttcctgt 90060 cagggccagctgagggacct catcctaacc tcccacttgg aggcacaatg ggctgtatcc 90120 ccccagcctggtttcctcgg cataacccgg atttaatgac cacagtgcct gatcacaggc 90180 atccttccctggaatcctgt tttcaaatgg cttgtctgtc caggaagttg ctcagtgcat 90240 gcgttagcagaagcggggag aaggagaaga gaccctggta ctgacgagct gggcttcccc 90300 ggtggggactgtgttctggg cacactccca ctctggctgg tagcagaaac acgggctttc 90360 atgtgcttggaatttgtgac tgtattgggg ttacctgagt gtcattgact ggtcccttca 90420 tgatgcggaaactgctaaat ttaaacaaaa aaaatccttc atttttctaa gaaagagaag 90480 aaagtcctttgcccaggacc tggataggga tatggtcatt attgtgtgaa gtgcaggact 90540 cataaaagcctattaagata atggctctaa ttcttctaag aaaaataatt cgatgggctg 90600 ttctttttaaacagctccct gaagtctatg tagtttgtaa tgcaggcttc caaggcctga 90660 gaggagcgcctagtgtattt ccccttcggt cccagtgggt gggagtgtcg aagtccagac 90720 aggacttgcattctggcatc atctcaggag cccctgagtc ccttgccact cgccttgcag 90780 actgtggtccagtatgtcag gactggggct caggaatctg cagggacagg tgcccaggag 90840 attctggccctggcgtgggg aagcatctat agaggaagtt gagcctcagc tttgattcag 90900 gtcctaacctggggctgtgg acagggcact cacctctcag cctcagggtc ctcactgcag 90960 agctagagtcagacatccca tctgtaggtc tgtgaaggct ggaggtaaag tgcatgaaca 91020 ggtggccagcagggtgctgg gcacgtggtt gatttttgat aggtgccact taaaattcct 91080 cctgacagtgccgctggcag ctggaggaag tagaagcttg gtactttgtg ggacactgac 91140 cactctgactggactttcct ttggcagagt ctttggattt gtggcccgga agcagggcag 91200 tgccacggataatgtgtgcc acctgtttgc agagcatgac cctgagcagc ctgccagtgc 91260 cattgtcaacttcgtatcaa aggtcatgat tggttcccca aagaaggtct gagaactccc 91320 ctccctccctggacccaccg atgcctctcg aagccctgga gacagccgtt gggtgagggt 91380 ggggcccccactttttacca aactagtaaa cctgacattc caggcccatg aggggaaaga 91440 ggatcttccagctctgcaaa aacaagaaca aacaacatca ccgtgaattg gcctttcctg 91500 aaagtgacttatctgacaca tctctgtagc cacatgcttt ttgggtagaa gaagctgggc 91560 atgggtgcaccccaccccct agggtcccca tgggaaaggg acatgcaagg aaacagcaca 91620 gaacacgaggtggtccccat gtccctggca cactagcatt ccgggggatg aggaatcccc 91680 agcccttgaggcagaggtgc cgagtgactg ccatgcttcg cccgtccgca tgggcgcttc 91740 tgtccagctgcacccgaggc cgggggtttc cctcacctcg gtcttcccaa gatggagatg 91800 ctaacgaaactgagaagggg gcgtatgttt gacgaaggtt tgtgcaagtc aggcccttct 91860 ggaacacagcagggcctaca acgaggggcc tttgcgatgg gctgtgagga tgggggtggt 91920 gggaagaattggccacgttg gagaccccat gccaccccac catggtgagt gctctgtgcc 91980 tcctgctcacctgtggtgag ctgggcgagc tgggcgagct gggcgagctg ggctggggag 92040 agcctgtgaggaccgagagg agaaatgaga agaaggaaca aaaatattat ttctatgtaa 92100 tttatattttacttatgcca aattatttat gataatttgc cattgctata ctgtaccagt 92160 gtcaaatgctgcagcctgcc aagctgtgat tttgtgaggc ttgtccctat gtaggatgca 92220 ccgcaggcccctggccactg aaagagtgtg cagtggactg tgggtctccc atatgcggtg 92280 ccgcccaaaggtggctttgc ctcaagcaac ctaccctgat gttttactca ttggaatgtt 92340 tttccccgattgtggatgac ttcttttctg atggagagag tccaggaggg atggaaaact 92400 cctggatttaagctcagcat cccccacatg ggcttttcga tcatcttcag gcctgaagct 92460 gcacgacctgaagttcgcct gcatttatca gccctctttg tgctgctcct tgccaccttg 92520 gggttcctgctggggaccat gtgtggttgt ggcatgtgtg agcagaaggg aggatgagga 92580 aaaagagaagaaaccccggt actgacaagc tgtttttgag tgccactgtt tgccatcatc 92640 taagccactgaatcaagtgt atttcaggct tatttcaaca ttccaatgcc ctggttttcc 92700 tgcttgaatctgttcgtggt caaaggtttg ggggaatttg tgaccctgga acatccccag 92760 agtgaaagatggagctgggc cacatcagaa taaggccttg gccccatcct ctcacagcct 92820 aggtgctctgcaggcatgct gactgtcctg attgcgatcc agcccgaaat tccctcctct 92880 gctttcaaaagtcaaatccc ccattcttag gccacactgg tgtcacaagc tcctgtcagg 92940 gagctggggtttgggaatgt gctttgtgaa ctctgcttta aagtgagggg ccgaggaaaa 93000 cttagaaacaggcagagttg gaagcagcca aatcacagtg ggtgttgtgt gtgtgtgcgt 93060 gtgtgcatgcgtgcgtgtat gcgtgtgtga aagcaggtgg accattccac tttttagctc 93120 ctattgatgcaccaaaccaa gtgcctcatt tctgtgccaa atgtttgcct tggtcgttgt 93180 ggacctccttctctaacttg cggtggcatg actgtcagga ggtgctggca ttttcagcag 93240 atcctcatgtgttgaccctg atgtctttag cagaggcctc tagcatctcg gtttttcatc 93300 cactgcaggaatgtggccac agggagcaga ggtttgtact ttccccaaga ggtcctcatc 93360 ctgagacggtctctacccat gtttaaccca aagagtgcag gccaggttcc ttatccttct 93420 gatgaaggatgagagagctc atttagaagt cagagcaaac tagggtctca gtattgagaa 93480 acgcagcctgccagggaatc acagagacat cggggtgccc gcgatggccc tcatgaagcc 93540 atgcctcgacggcattcagg aagccctgca aacgtgcttt ttgaactcat tggccaggtg 93600 tgatttttacacaaggtaaa cgtggtcaag ggcatcgggg aatttgctcc aagcagatag 93660 ctccctctgaggaaccaaag gaagcaagtt tccacgattt ctgaagagct ggtataggaa 93720 gtttctttcttccttttgtg ttacatgtgc attaaacaga acaagctgtg tgtcatcaca 93780 gattgtactgtgggctcaga aaccgtgaga gagcccccac cgtggacacc ggctctaggg 93840 ccacaggaaaaggaacgttt ccaggcattt tgtctccagg gctcccgctg gacaggcacg 93900 tactgccctggggagtaaat gcggagagtt cacgaactgt gcccaacgca tgttatagcc 93960 agggtcctactaactactca gtaaaagaac gtattgttgt attcctccag tgttaagcta 94020 tagccatgttaaaagtcact gtgcatttat tctcagcatc aaataccttg taacgtcttc 94080 tctgccttgttagtgcatat ttttactttt ctgatactgt aaagaatata tccagtatgt 94140 aaatgaatgttctataaatc ttttgtatag tcattttctc tgctccttaa atatcatctc 94200 tattcagagtataataaaat tatgaacttg gtaagcctcc gtgagacggt atttgtgtac 94260 cctgaagtatgtttgtgtgt gtttgcacat gtgtgtatgc gtgtgcatgt gtgcactttt 94320 gtgtatgtgtctctgcaaat gtgtgcatgt gcttgcacag atgtatcttt gtccacatgt 94380 gtctgtgcgtctgcgtgtgt gctcgttcat gaatgtgtgt ctgtgtgttt acaaatcagt 94440 ggattcagttataaaatcac aaatgttttg ctgtcaggaa tatattaaga tttctgccca 94500 gagacctttgaaatgttata ttcaaagatc tttgaagatt aaccacaatg cagtgttgat 94560 aatgggtcctgacatgtttt cgatgttgct atgaataccc ctacacacac actggctgca 94620 gaaacacacccctgaagtgg gccagctcct gaaggacctc ccagtgtcta ttgaagtggg 94680 cactaaaagtgaatattgag actggtggat tcacttacac atcaacagat gctggagaca 94740 gccagtgtcacctgcctctt ctgttccccc agccaaggct cctccctccc caccttaagg 94800 gagtttgcccttttctacgg actctgccct cttgggcctg tcccctccct cctgagtcag 94860 gtgatgggacacaggacccc atctaacctc actccctcag atccaaacca gaaaagaagc 94920 aatgctttgggctcatttgg ttatggacag ataaagatgg cagttagctg ccaccctgtg 94980 cacagttttctgcctgcgtg g 95001 12 20 DNA Artificial Sequence AntisenseOligonucleotide 12 tcagcttcct gagtctcccg 20 13 20 DNA ArtificialSequence Antisense Oligonucleotide 13 agcatcgttg tcgtactgcc 20 14 20 DNAArtificial Sequence Antisense Oligonucleotide 14 ccatgcacat ttggagaagg20 15 20 DNA Artificial Sequence Antisense Oligonucleotide 15 cctttccccatgcacatttg 20 16 20 DNA Artificial Sequence Antisense Oligonucleotide 16gggtcatcgt ctctttgacg 20 17 20 DNA Artificial Sequence AntisenseOligonucleotide 17 taaaattctg ccatcgataa 20 18 20 DNA ArtificialSequence Antisense Oligonucleotide 18 tctggagaca caggaaatgc 20 19 20 DNAArtificial Sequence Antisense Oligonucleotide 19 gctgttttca catacggtgt20 20 20 DNA Artificial Sequence Antisense Oligonucleotide 20 gggcagcttcgtggttcacg 20 21 20 DNA Artificial Sequence Antisense Oligonucleotide 21gcatcgtgga tttgggtccg 20 22 20 DNA Artificial Sequence AntisenseOligonucleotide 22 gcctgctgaa aggagggcgt 20 23 20 DNA ArtificialSequence Antisense Oligonucleotide 23 tggaaatggt gcaggaagaa 20 24 20 DNAArtificial Sequence Antisense Oligonucleotide 24 gtccttcctg gtaccggaga20 25 20 DNA Artificial Sequence Antisense Oligonucleotide 25 gggacagctcatggctgtgg 20 26 20 DNA Artificial Sequence Antisense Oligonucleotide 26agcaaggccc ccaggtcctc 20 27 20 DNA Artificial Sequence AntisenseOligonucleotide 27 ggacaggaag ccattgtcgg 20 28 20 DNA ArtificialSequence Antisense Oligonucleotide 28 tggtggctgc tgtgtccagg 20 29 20 DNAArtificial Sequence Antisense Oligonucleotide 29 ctgtgcgggc tggagaagcc20 30 20 DNA Artificial Sequence Antisense Oligonucleotide 30 aggccgcttctgaagccttg 20 31 20 DNA Artificial Sequence Antisense Oligonucleotide 31atggcgatgg cttgttctct 20 32 20 DNA Artificial Sequence AntisenseOligonucleotide 32 cgagttcatt ggccaaatct 20 33 20 DNA ArtificialSequence Antisense Oligonucleotide 33 tatggttcat tcgagcaccc 20 34 20 DNAArtificial Sequence Antisense Oligonucleotide 34 cggcaaggcc aagggcgtga20 35 20 DNA Artificial Sequence Antisense Oligonucleotide 35 tccaatggatctctctctgg 20 36 20 DNA Artificial Sequence Antisense Oligonucleotide 36ctccacagag ttcaagtacc 20 37 20 DNA Artificial Sequence AntisenseOligonucleotide 37 tgatgctcag ggccttctgg 20 38 20 DNA ArtificialSequence Antisense Oligonucleotide 38 ggtaatgcct ccggaagaag 20 39 20 DNAArtificial Sequence Antisense Oligonucleotide 39 catctttgat ccacttcctg20 40 20 DNA Artificial Sequence Antisense Oligonucleotide 40 actggcaggctgctcagggt 20 41 20 DNA Artificial Sequence Antisense Oligonucleotide 41gggagttctc agaccttctt 20 42 20 DNA Artificial Sequence AntisenseOligonucleotide 42 acggctgtct ccagggcttc 20 43 20 DNA ArtificialSequence Antisense Oligonucleotide 43 tgtcagataa gtcactttca 20 44 20 DNAArtificial Sequence Antisense Oligonucleotide 44 gcttcttcta cccaaaaagc20 45 20 DNA Artificial Sequence Antisense Oligonucleotide 45 atggcagtcactcggcacct 20 46 20 DNA Artificial Sequence Antisense Oligonucleotide 46gggtgcagct ggacagaagc 20 47 20 DNA Artificial Sequence AntisenseOligonucleotide 47 tacgccccct tctcagtttc 20 48 20 DNA ArtificialSequence Antisense Oligonucleotide 48 gggcctgact tgcacaaacc 20 49 20 DNAArtificial Sequence Antisense Oligonucleotide 49 ctaacgtggc caattcttcc20 50 20 DNA Artificial Sequence Antisense Oligonucleotide 50 gaggcacagagcactcacca 20 51 20 DNA Artificial Sequence Antisense Oligonucleotide 51aggctgcagc atttgacact 20 52 20 DNA Artificial Sequence AntisenseOligonucleotide 52 cctgcggtgc atcctacata 20 53 20 DNA ArtificialSequence Antisense Oligonucleotide 53 cacagtccac tgcacactct 20 54 20 DNAArtificial Sequence Antisense Oligonucleotide 54 caatgagtaa aacatcaggg20 55 20 DNA Artificial Sequence Antisense Oligonucleotide 55 gggaaaaacattccaatgag 20 56 20 DNA Artificial Sequence Antisense Oligonucleotide 56gaagatgatc gaaaagccca 20 57 20 DNA Artificial Sequence AntisenseOligonucleotide 57 ccacacatgg tccccagcag 20 58 20 DNA ArtificialSequence Antisense Oligonucleotide 58 cactcaaaaa cagcttgtca 20 59 20 DNAArtificial Sequence Antisense Oligonucleotide 59 gtcacaaatt cccccaaacc20 60 20 DNA Artificial Sequence Antisense Oligonucleotide 60 caggacagtcagcatgcctg 20 61 20 DNA Artificial Sequence Antisense Oligonucleotide 61cagagttcac aaagcacatt 20 62 20 DNA Artificial Sequence AntisenseOligonucleotide 62 cactgtgatt tggctgcttc 20 63 20 DNA ArtificialSequence Antisense Oligonucleotide 63 aagtggaatg gtccacctgc 20 64 20 DNAArtificial Sequence Antisense Oligonucleotide 64 atgaggatct gctgaaaatg20 65 20 DNA Artificial Sequence Antisense Oligonucleotide 65 ccgagatgctagaggcctct 20 66 20 DNA Artificial Sequence Antisense Oligonucleotide 66gctctgactt ctaaatgagc 20 67 20 DNA Artificial Sequence AntisenseOligonucleotide 67 tggcaggctg cgtttctcaa 20 68 20 DNA ArtificialSequence Antisense Oligonucleotide 68 attccctggc aggctgcgtt 20 69 20 DNAArtificial Sequence Antisense Oligonucleotide 69 tcaaaaagca cgtttgcagg20 70 20 DNA Artificial Sequence Antisense Oligonucleotide 70 ggagctatctgcttggagca 20 71 20 DNA Artificial Sequence Antisense Oligonucleotide 71acgttccttt tcctgtggcc 20 72 20 DNA Artificial Sequence AntisenseOligonucleotide 72 tgaactctcc gcatttactc 20 73 20 DNA ArtificialSequence Antisense Oligonucleotide 73 ctggctataa catgcgttgg 20 74 20 DNAArtificial Sequence Antisense Oligonucleotide 74 aatacaacaa tacgttcttt20 75 20 DNA Artificial Sequence Antisense Oligonucleotide 75 atggctatagcttaacactg 20 76 20 DNA Artificial Sequence Antisense Oligonucleotide 76acagtgactt ttaacatggc 20 77 20 DNA Artificial Sequence AntisenseOligonucleotide 77 tgagaataaa tgcacagtga 20 78 20 DNA ArtificialSequence Antisense Oligonucleotide 78 atatattctt tacagtatca 20 79 20 DNAArtificial Sequence Antisense Oligonucleotide 79 tttatagaac attcatttac20 80 20 DNA Artificial Sequence Antisense Oligonucleotide 80 atactctgaatagagatgat 20 81 20 DNA Artificial Sequence Antisense Oligonucleotide 81ccaagttcat aattttatta 20 82 20 DNA Artificial Sequence AntisenseOligonucleotide 82 ccttcagccc cactgaagta 20 83 20 DNA ArtificialSequence Antisense Oligonucleotide 83 tcattcttac catacggtgt 20 84 20 DNAArtificial Sequence Antisense Oligonucleotide 84 gatttcttac cacgtcctcc20 85 20 DNA Artificial Sequence Antisense Oligonucleotide 85 tgctgaaaaactcctataca 20 86 20 DNA Artificial Sequence Antisense Oligonucleotide 86ctttcactgc ctgatgccca 20 87 20 DNA Artificial Sequence AntisenseOligonucleotide 87 actaatgatg ttgaacatct 20 88 20 DNA ArtificialSequence Antisense Oligonucleotide 88 tttgatccac cttgcaaatt 20 89 20 DNAArtificial Sequence Antisense Oligonucleotide 89 tccaaagact ctgccaaagg20 90 20 DNA H. sapiens 90 cgggagactc aggaagctga 20 91 20 DNA H. sapiens91 ggcagtacga caacgatgct 20 92 20 DNA H. sapiens 92 ccttctccaaatgtgcatgg 20 93 20 DNA H. sapiens 93 ttatcgatgg cagaatttta 20 94 20 DNAH. sapiens 94 cgtgaaccac gaagctgccc 20 95 20 DNA H. sapiens 95cggacccaaa tccacgatgc 20 96 20 DNA H. sapiens 96 ttcttcctgc accatttcca20 97 20 DNA H. sapiens 97 tctccggtac caggaaggac 20 98 20 DNA H. sapiens98 ccacagccat gagctgtccc 20 99 20 DNA H. sapiens 99 gaggacctgggggccttgct 20 100 20 DNA H. sapiens 100 cctggacaca gcagccacca 20 101 20DNA H. sapiens 101 ggcttctcca gcccgcacag 20 102 20 DNA H. sapiens 102caaggcttca gaagcggcct 20 103 20 DNA H. sapiens 103 agatttggcc aatgaactcg20 104 20 DNA H. sapiens 104 gggtgctcga atgaaccata 20 105 20 DNA H.sapiens 105 tcacgccctt ggccttgccg 20 106 20 DNA H. sapiens 106ccagagagag atccattgga 20 107 20 DNA H. sapiens 107 ggtacttgaa ctctgtggag20 108 20 DNA H. sapiens 108 ccagaaggcc ctgagcatca 20 109 20 DNA H.sapiens 109 accctgagca gcctgccagt 20 110 20 DNA H. sapiens 110aagaaggtct gagaactccc 20 111 20 DNA H. sapiens 111 gaagccctgg agacagccgt20 112 20 DNA H. sapiens 112 tgaaagtgac ttatctgaca 20 113 20 DNA H.sapiens 113 gctttttggg tagaagaagc 20 114 20 DNA H. sapiens 114aggtgccgag tgactgccat 20 115 20 DNA H. sapiens 115 gcttctgtcc agctgcaccc20 116 20 DNA H. sapiens 116 gaaactgaga agggggcgta 20 117 20 DNA H.sapiens 117 ggtttgtgca agtcaggccc 20 118 20 DNA H. sapiens 118ggaagaattg gccacgttag 20 119 20 DNA H. sapiens 119 tggtgagtgc tctgtgcctc20 120 20 DNA H. sapiens 120 agtgtcaaat gctgcagcct 20 121 20 DNA H.sapiens 121 tatgtaggat gcaccgcagg 20 122 20 DNA H. sapiens 122ccctgatgtt ttactcattg 20 123 20 DNA H. sapiens 123 ctcattggaa tgtttttccc20 124 20 DNA H. sapiens 124 tgggcttttc gatcatcttc 20 125 20 DNA H.sapiens 125 ctgctgggga ccatgtgtgg 20 126 20 DNA H. sapiens 126tgacaagctg tttttgagtg 20 127 20 DNA H. sapiens 127 ggtttggggg aatttgtgac20 128 20 DNA H. sapiens 128 caggcatgct gactgtcctg 20 129 20 DNA H.sapiens 129 gaagcagcca aatcacagtg 20 130 20 DNA H. sapiens 130gcaggtggac cattccactt 20 131 20 DNA H. sapiens 131 cattttcagc agatcctcat20 132 20 DNA H. sapiens 132 agaggcctct agcatctcgg 20 133 20 DNA H.sapiens 133 gctcatttag aagtcagagc 20 134 20 DNA H. sapiens 134ttgagaaacg cagcctgcca 20 135 20 DNA H. sapiens 135 aacgcagcct gccagggaat20 136 20 DNA H. sapiens 136 cctgcaaacg tgctttttga 20 137 20 DNA H.sapiens 137 tgctccaagc agatagctcc 20 138 20 DNA H. sapiens 138ggccacagga aaaggaacgt 20 139 20 DNA H. sapiens 139 ccaacgcatg ttatagccag20 140 20 DNA H. sapiens 140 cagtgttaag ctatagccat 20 141 20 DNA H.sapiens 141 gccatgttaa aagtcactgt 20 142 20 DNA H. sapiens 142tcactgtgca tttattctca 20 143 20 DNA H. sapiens 143 tgatactgta aagaatatat20 144 20 DNA H. sapiens 144 atcatctcta ttcagagtat 20 145 20 DNA H.sapiens 145 tacttcagtg gggctgaagg 20 146 20 DNA H. sapiens 146tgtataggag tttttcagca 20 147 20 DNA H. sapiens 147 tgggcatcag gcagtgaaag20 148 20 DNA H. sapiens 148 agatgttcaa catcattagt 20 149 20 DNA H.sapiens 149 cctttggcag agtctttgga 20

What is claimed is:
 1. A compound 8 to 80 nucleobases in length targetedto a nucleic acid molecule encoding hypothetical tumor endothelialmarker, wherein said compound specifically hybridizes with said nucleicacid molecule encoding hypothetical tumor endothelial marker andinhibits the expression of hypothetical tumor endothelial marker.
 2. Thecompound of claim 1 which is an antisense oligonucleotide.
 3. Thecompound of claim 2 wherein the antisense oligonucleotide comprises atleast one modified internucleoside linkage.
 4. The compound of claim 3wherein the modified internucleoside linkage is a phosphorothioatelinkage.
 5. The compound of claim 2 wherein the antisenseoligonucleotide comprises at least one modified sugar moiety.
 6. Thecompound of claim 5 wherein the modified sugar moiety is a2′-O-methoxyethyl sugar moiety.
 7. The compound of claim 2 wherein theantisense oligonucleotide comprises at least one modified nucleobase. 8.The compound of claim 7 wherein the modified nucleobase is a5-methylcytosine.
 9. The compound of claim 2 wherein the antisenseoligonucleotide is a chimeric oligonucleotide.
 10. A compound 8 to 80nucleobases in length which specifically hybridizes with at least an8-nucleobase portion of a preferred target region on a nucleic acidmolecule encoding hypothetical tumor endothelial marker.
 11. Acomposition comprising the compound of claim 1 and a pharmaceuticallyacceptable carrier or diluent.
 12. The composition of claim 11 furthercomprising a colloidal dispersion system.
 13. The composition of claim11 wherein the compound is an antisense oligonucleotide.
 14. A method ofinhibiting the expression of hypothetical tumor endothelial marker incells or tissues comprising contacting said cells or tissues with thecompound of claim 1 so that expression of hypothetical tumor endothelialmarker is inhibited.
 15. A method of treating an animal having a diseaseor condition associated with hypothetical tumor endothelial markercomprising administering to said animal a therapeutically orprophylactically effective amount of the compound of claim 1 so thatexpression of hypothetical tumor endothelial marker is inhibited. 16.The method of claim 15 wherein the disease or condition is ahyperproliferative disorder.
 17. The method of claim 16 wherein thehyperproliferative disorder is cancer.
 18. The method of claim 17wherein the cancer is colon cancer.
 19. The method of claim 15 whereinthe disease or condition arises from aberrant angiogenesis.
 20. A methodof screening for an antisense compound, the method comprising the stepsof: a. contacting a preferred target region of a nucleic acid moleculeencoding hypothetical tumor endothelial marker with one or morecandidate antisense compounds, said candidate antisense compoundscomprising at least an 8-nucleobase portion which is complementary tosaid preferred target region, and b. selecting for one or more candidateantisense compounds which inhibit the expression of a nucleic acidmolecule encoding hypothetical tumor endothelial marker.